Pestivirus

ABSTRACT

The present invention relates to a novel porcine pestivirus, to proteins of the virus and to vaccines based upon the virus and proteins thereof. The invention also relates to DNA fragments comprising a gene of the virus and to DNA vaccines based upon genes of the virus. Furthermore the invention relates to antibodies that are reactive with the novel virus and to diagnostic tests for the detection of the virus or antibodies against the virus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage entry under 35 U.S.C. § 371 ofPCT/EP2015/080400 filed on Dec. 18, 2015, which claims priority to EP14199430.1, filed on Dec. 19, 2014. The content of PCT/EP2015/080400 ishereby incorporated by reference in its entirety.

The present invention relates to a novel pestivirus, to proteins of thevirus and to vaccines based upon the virus and proteins thereof. Theinvention also relates to DNA fragments comprising a gene of the virusand to DNA vaccines based upon genes of the virus. Furthermore theinvention relates to antibodies that are reactive with the novel virusand to diagnostic tests for the detection of the virus or antibodiesagainst the virus.

Over the last decades, world-wide a strong increase is seen in theconsumption of pig meat. As a consequence, an increase is seen in thenumber and the size of farms, in order to meet the increasing needs ofthe market. As is known from animal husbandry in general, large numbersof animals living closely together are vulnerable to known diseases andto diseases hardly known or seen or even unknown before the days oflarge-scale commercial farming.

One disease for which the causative agent awaits identification is knownto exist already since the early 20^(th) century, when “dancing pigs”were mentioned by Kinsley in Veterinary Medicine 1922; 17. Over thecourse of nearly a century several articles have been published thatdescribe the same symptoms under varying names, including “shaking pigdisease”, tremor in pigs, Myoclonia Congenita ⁽¹⁾ or congenital tremor(CT)⁽²⁾. The disease will further be referred to as CT. Symptoms of CTare tremors of the head and legs of newborn pigs that vary in severitybut are absent during sleep. These tremors can be aggravated byexcitement and cold. They last for several weeks to months but decreaseas the pigs grow older. Although the shaking itself does not directlycause death the tremors can prevent the piglets from finding a teat tosuckle. This can then result in death by starvation. The disease iswidespread and occurs regularly in pig farms all over the world.

Several conditions are known to cause CT, and currently these conditionsare classified in two groups; A and B. Group A consists of the caseswith visible histological lesions, whereas the cases of Group B displayno apparent lesions. Group A is further divided into five subgroups,based on the different causes of CT. Group A-I cases of CT are known tobe caused by Classical Swine Fever (CSF) virus. The cause of Group A-IIICT is a genetic (sex-linked) defect existing only in the Landrace breed,while a recessive genetic (autosomal-linked) defect in the Saddlebackbreed is the cause of type A-IV. Group A-V cases are caused bytrichlorfon toxicosis, an intoxication which is often linked toorganophosphorus treated food^((3, 4)).

Group A-II cases have been, and still are, the most puzzling cases. Theyare suspected to be caused by an unknown infectious agent.

Although Group A-II CT has been associated with PCV infection in thepast⁽⁵⁾, various studies have now demonstrated the opposite. Forexample, PCV is absent in neuronal tissue of pigs with CT⁽⁶⁾ and only asmall, insignificant, amount of PCV was found in non-neuronal tissue⁽⁴⁾.All in all no conclusive evidence exists so far for the cause of GroupA-II CT. There is sufficient reason to believe, however, that Group A-IICT is caused by an infectious agent. Most of the Group A-II shakingpiglets are born into the litters of gilts (i.e. female pigs in theperiod between fertilisation and their first litter) that have recentlybeen introduced into a new environment. Remarkably, after a first litterwith shaking piglets subsequent litters of the same sow hardly ever showsigns of CT. This is an indication that some kind of immunity developsin the sow, protecting against the agent that causes CT. Some 40 yearsago, Patterson et al. (50) managed to induce Group A-II CT in pigletsthrough experimental infection of pregnant sows with an emulsion ofspinal cord, brains and spleens of clinically affected pigs.

But as indicated above, no causative infectious agent has ever beenisolated from CT piglets nor from pregnant sows.

It is an objective of the present invention to provide a new infectiousagent that is the causative agent of Group A-II CT, as well as vaccinesaiming at combating the disease. Moreover, it is an objective of thepresent invention to provide means to detect and identify thedisease-associated infectious agent.

In order to finally detect and isolate the causative agent of Group A-IICT, sera and in many cases additional biological material of pigletssuffering from Group A-II CT were obtained from September of 2012 untilearly 2014, on 8 different farms in the Netherlands. These 8 farms had ahistory of CT-piglets (Typically in one out of four litters, piglets arefound suffering from CT during an epidemic peak on one specific farm).

A pig farm in the Netherlands was diagnosed with an outbreak ofcongenital tremor type A-II in early 2012. Piglets born from gilt, firstparity animals, were primarily affected but also higher parity sows wereoccasionally affected. Diagnosis was based on clinical observations andsubsequent exclusion of congenital tremor types A-I, A-III, A-IV and A-Vas the possible cause for disease. Clinically affected piglets showedtremor in different grades, due to excessive muscle contractions duringactivity. The symptoms diminished when sleeping. Piglet loss was asecondary effect caused by the inability of affected animals to feedthemselves, especially during the first week after birth.Histologically, the brain and the spinal cord were characterized byhypomyelinization. (Histological abnormalities are however not alwaysseen in affected piglets. In the literature, the extent ofhypomyelinization is also described as being variable). As furtherdescribed below, not all affected pigs survived. In those that survived,the tremor diminished and finally disappeared as pigs grew older. In thefirst 20 weeks of the year 2012, a total of 48 litters with symptoms ofcongenital tremor were born from gilts, out of 231 μlitters born fromgilts in total. This equals 21% of all litters born from gilts. At thepeak of infection, 8 weeks after the initial outbreak, 85% of the giltlitters showed piglets with congenital tremor type A-II. The percentagepiglet loss (piglet death) till weaning was 26% in affected litters,compared to 11% in non-affected litters. In affected litters, 60% ofpiglet death was attributable to congenital tremor. The total number ofpiglets born per litter was not affected. Congenital tremor affectedboth newborn male and female piglets, and prevalence within the littervaried between <10%-100%.

Problems with outbreaks of congenital tremor have continued on this farmsince 2012, and affected piglets were obtained in 2013 and 2014 (seebelow). However, the incidence rate decreased.

Blood plasma samples were obtained in March 2012 (6 samples, all pigletswith symptoms of CT where non-A-II causes could be excluded) and April2012 (5 samples, all piglets with symptoms of CT where non-A-II causescould be excluded). A new virus, temporarily called “Michael” (M) wasdetected in 11/11 samples.

More blood plasma samples were obtained from the same farm in July 2012.A total of 16 serum samples from piglets born from 2 sows and 1 giltwere analyzed. None of these piglets showed congenital tremor. Michael 1was found in 1/16 samples.

A new outbreak of the disease was diagnosed in January 2013. Fournewborn pre-colostral piglets were obtained for necropsy, all showedsymptoms of CT where non-A-II causes could be excluded. The new viruswas named Michael 1A because, although it originated from the same farm,significant time had elapsed between the original outbreak and theoccurrence of new clinical problems. The new virus Michael 1A wasdetected in 4/4 piglets.

Again a new outbreak of the disease was diagnosed in March 2013. Threenewborn pre-colostral piglets were obtained for necropsy, all showedsymptoms of CT where non-A-II causes could be excluded. This virus wasnamed Michael 1B (M1B). The new virus Michael 1B was detected in 3/3samples. Brains and spinal cord showed signs of demyelinization (seeFIG. 2).

A new outbreak of the disease was diagnosed in January 2014. Fournewborn pre-colostral piglets were obtained, all showing symptoms of CTwhere non-A-II causes could be excluded. This virus was named Michael 1C(M1C). The new virus Michael 1C was detected in 4/4 samples. Necropsy onan additional 3 piglets was performed in February 2014, again all 3piglets showed Group A-II CT, and Michael was detected in 3/3 samples.

Post mortem examination was performed on piglets from outbreaks inJanuary 2013, March 2013 and February 2014. Brains and spinal cordshowed signs of demyelinization.

A total of 7 sera obtained from newborn pre-colostral piglets from afarm with no history of congenital tremor type A-II was used as negativecontrol for PCR and for post mortem examination. All plasma samples werenegative for Michael virus, and no pathological abnormalities wereobserved in these piglets.

Comparable analysis of Group A-II CT outbreaks was done on 7 other farmsin the Netherlands. Samples of CT-litters were analysed and the novelvirus was found in 100% of the CT-piglets from which pre-colostralmaterial was taken (material taken before the first ingestion ofcolostrum or mother milk).

The novel virus according to the invention is not yet officiallyclassified, but for the moment the virus is best referred to as “GroupA-II congenital tremor associated porcine pestivirus”. The virus willalso be referred to as CTAPV below.

The sequence of the viral genome was analysed and revealed that thenovel virus unexpectedly bears some albeit a relatively low level ofresemblance to the family of Flaviviridae, more specifically to thegenus Pestivirus within this family. Known members of the genusPestivirus are Classical Swine Fever virus, Bovine Viral Diarrhea virusand Border Disease virus.

Pestivirus virions are about 50 nm in diameter, spherical and enveloped,and they comprise a single stranded positive-sense RNA which is around12 kilobases (kb) long.

The full length DNA sequence of a representative of the new virus ispresented in SEQ ID NO: 19.

The genetic organization of the novel virus closely follows that of theknown pestiviruses (see FIG. 1). The pestivirus genome encodes a singlepolyprotein NH2-C-E^(rns)-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH thatis processed co- and post-translationally into both structural proteins(“Core” protein (C), and proteins E^(rns), E1 and E2) and non-structural(NS) proteins. The amino-terminal part of the polyprotein is cleaved byhost cell proteases and its cleavage products, core and envelope(E^(rns), E1 and E2) proteins are believed to be the major constituentsof pestivirus particles (virions).

The structural protein E^(rns), also known as E0 or gp44/48 is anenvelope protein with the unique property of having RNase activity (12).It is secreted from infected cells in a relatively large amount (13).However, an even larger amount remains membrane bound (14). One of theroles of E^(rns) appears to be in interfering with the host immunesystem by inhibiting the interferon response using its RNase activity(15). Such a role in virulence is further supported by the fact thatviral strains that are missing E^(rns) become attenuated (16). E1 andE2, previously known as gp33 and gp55 (and previously confusingly alsoas E1), respectively, are the other two envelope glycoproteins. Thestructural protein E2 forms homodimers and heterodimers with E1 (17,18). Especially heterodimers of E1 and E2 protein are important forpestiviruses to enter their host, whereas E^(rns) does not seem to berequired for virus entry (19, 20). Neutralizing antibodies primarilytarget E^(rns) and E2, and to a lesser extend to E1 (17, 21).

The gene encoding the envelope protein E^(rns) consisting of 216 aminoacids is found at position 1258-1899 of SEQ ID NO: 19 and the geneencoding the envelope protein E2 consisting of 211 amino acids is foundat position 2479-3111 of SEQ ID NO: 19. The gene encoding the envelopeprotein E1 consisting of 193 amino acids is found at position 1900-2478of SEQ ID NO: 19.

An example of the DNA sequence of the gene encoding the envelope proteinE^(rns) is depicted in SEQ ID NO: 1. SEQ ID NO: 2 represents the aminoacid sequence of the E^(rns) protein.

An example of the DNA sequence of the gene encoding the envelope proteinE2 is depicted in SEQ ID NO: 3. SEQ ID NO: 4 represents the amino acidsequence of the E2 protein.

An example of the DNA sequence of the gene encoding the envelope proteinE1 is depicted in SEQ ID NO: 5. SEQ ID NO: 6 represents the amino acidsequence of the E1 protein.

The full sequences of the novel virus was used to make phylogenetictrees based on the Maximum Likelihood method, the Poisson correctionmodel and bootstrap analysis (500 replicates).

These trees were made using the program MEGA, version 5, using standardsettings. (MEGA5: Molecular Evolutionary Genetics Analysis Using MaximumLikelihood, Evolutionary Distance, and Maximum Parsimony Methods.Koichiro Tamura, Daniel Peterson, Nicholas Peterson, Glen Stecher,Masatoshi Nei and Sudhir Kumar. Mol. Biol. Evol. 28(10): 2731-2739. 2011doi:10.1093/molbev/msr121 Advance Access publication May 4, 2011).

The phylogenetic tree based upon the whole sequence of the novelpestivirus is presented in FIG. 3. The percentage bootstrap support isspecified at the nodes. Distance bars indicate the number of nucleotidesubstitutions per site.

It is clear from FIG. 3, that whereas the pestiviruses Border Diseasevirus, pestivirus of reindeer, classical swine fever virus, bovine viraldiarrhea virus, pestivirus of giraffe and Bungowannah virus arerelatively closely related, the novel virus according to the inventionis more distantly related to each of these viruses.

In FIG. 4, a phylogenic tree is presented wherein 10 different isolatesof the virus according to the invention are compared.

It can be seen that the isolates M1, M1A, M1B and M1C (SEQ ID NO: 19,20, 21, 22), isolated on the same farm, but over three years, are themost closely related to each other. Isolates from other farms show asomewhat greater variation. M2, M4 and M9 (SEQ ID NO: 23, 25, 29) aremore related to each other than to the M1 group. The same is true forboth M3, M6 and M8 (SEQ ID NO: 24, 26, 28). M7 (SEQ ID NO: 27) is notincluded. This indicates that there are small genetic changes betweenisolates. This is to be expected for RNA viruses, and this observationis in line with what is seen for other pestiviruses.

SEQ ID NO: 1, 3 and 5 show typical examples of the nucleotide sequenceof the genes encoding E^(rns), E2 and E1 of a virus according to theinvention respectively.

SEQ ID NO: 2, 4 and 6 show typical examples of the amino acid sequenceof an E^(rns), E2 and E1 protein of a virus according to the inventionrespectively.

It will be understood that for these proteins natural variations canexist between individual representatives of the Group A-II congenitaltremors-associated virus. Genetic variations leading to minor changes ine.g. the E^(rns), E2 and E1 amino acid sequence do exist. First of all,there is the so-called “wobble in the second and third base” explainingthat nucleotide changes may occur that remain unnoticed in the aminoacid sequence they encode: e.g. triplets TTA, TTG, TCA, TCT, TCG and TCCall encode Leucine. In addition, minor variations betweenrepresentatives of the novel porcine pestivirus according to theinvention may be seen in amino acid sequence. These variations can bereflected by (an) amino acid difference(s) in the overall sequence or bydeletions, substitutions, insertions, inversions or additions of (an)amino acid(s) in said sequence. Amino acid substitutions which do notessentially alter biological and immunological activities, have beendescribed, e.g. by Neurath et al in “The Proteins” Academic Press NewYork (1979). Amino acid replacements between related amino acids orreplacements which have occurred frequently in evolution are, interalia, Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M. D.,Atlas of protein sequence and structure, Nat. Biomed. Res. Found.,Washington D.C., 1978, vol. 5, suppl. 3). Other amino acid substitutionsinclude Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Thr/Phe,Ala/Pro, Lys/Arg, Leu/Ile, Leu/Val and Ala/Glu. Based on thisinformation, Lipman and Pearson developed a method for rapid andsensitive protein comparison (Science 227, 1435-1441, 1985) anddetermining the functional similarity between homologous proteins. Suchamino acid substitutions of the exemplary embodiments of this invention,as well as variations having deletions and/or insertions are within thescope of the invention.

This explains why E^(rns), E2 and E1, when isolated from differentrepresentatives of a porcine pestivirus according to the invention, mayhave homology levels that are significantly below 100%, while stillrepresenting the E^(rns), E2 and E1 of the novel pestivirus according tothe invention.

This is clearly reflected e.g. in the phylogenetic tree for thepestiviral gene N^(pro) in Becher, P. et al.⁽⁴⁹⁾, where it is shown thathighly related pestiviruses nevertheless have significantly differentoverall genomic nucleotide sequences as well as significantly differentN^(pro) gene nucleotide sequences.

Thus, a first embodiment of the present invention relates to an isolatedvirus which is a member of the pestiviruses, wherein the virus ischaracterized in that

a) the virus is the causative agent of Group A-II congenital tremors inpigs and

b) the virus has a viral genome comprising a gene encoding an envelopeprotein E^(rns), a gene encoding an envelope protein E2 and a geneencoding an envelope protein E1, wherein the nucleotide sequence of theE^(rns) gene has a level of identity of at least 80% to the nucleotidesequence as depicted in SEQ ID NO: 1 and/or the nucleotide sequence ofthe E2 gene has a level of identity of at least 80% to the nucleotidesequence as depicted in SEQ ID NO: 3 and/or the nucleotide sequence ofthe E1 gene has a level of identity of at least 80% to the nucleotidesequence as depicted in SEQ ID NO: 5.

For the purpose of this invention, a level of identity is to beunderstood as the percentage of identity between e.g. the sequence ofSEQ ID NO: 1 and the corresponding region encoding the E^(rns) of apestivirus of which the level of identity is to be determined.

A suitable program for the determination of a level of identity is thenucleotide blast program (blastn) of NCBI's Basic Local Alignment SearchTool, using the “Align two or more sequences” option and standardsettings (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

For the purpose of this invention, isolated means: set free from tissuewith which the virus is associated in nature. An example of an isolatedvirus is the virus as present in cell culture.

A preferred form of this embodiment relates to such a virus that has anE^(rns) gene that has a level of identity of at least 82%, morepreferably 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or even 100%, in that order of preference, to the nucleotidesequence of the E^(rns) as depicted in SEQ ID NO: 1.

Another preferred form of this embodiment relates to such a virus thathas an E2 gene that has a level of identity of at least 82%, morepreferably 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or even 100%, in that order of preference, to the nucleotidesequence of the E2 gene as depicted in SEQ ID NO: 3.

Again another preferred form of this embodiment relates to such a virusthat has an E1 gene that has a level of identity of at least 82%, morepreferably 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or even 100%, in that order of preference, to the nucleotidesequence of the E1 gene as depicted in SEQ ID NO: 5.

A more preferred form of this embodiment relates to an isolated viruswhich is a member of the pestiviruses, said virus being characterized inthat

a) the virus is the causative agent of Group A-II congenital tremors inpigs and

b) the virus has a viral genome comprising a gene encoding an envelopeprotein E^(rns), a gene encoding an envelope protein E2 and a geneencoding an envelope protein E1, wherein the nucleotide sequence of theE^(rns) gene has a level of identity of at least 80% to the nucleotidesequence as depicted in SEQ ID NO: 1 and the nucleotide sequence of theE2 gene has a level of identity of at least 80% to the nucleotidesequence as depicted in SEQ ID NO: 3 and the nucleotide sequence of theE1 gene has a level of identity of at least 80% to the nucleotidesequence as depicted in SEQ ID NO: 5.

Another, alternative, way to characterize the virus according to theinvention depends on a PCR-test using primer sets that are specific forthe NS5B gene sequence or the 5′UTR sequence of a virus according to theinvention.

An overview of the various primers and the size of the PCR products madeusing these primers are represented in table a and b.

Four different primer sets of which the sequence is depicted in SEQ IDNO: 7-8, SEQ ID NO: 9-10, SEQ ID NO: 11-12 and SEQ ID NO: 13-14 wereelected for their specificity for the NS5B region of the virus. ThePCR-tests using the first primer set (SEQ ID NO: 7-8), the second primerset (SEQ ID NO: 9-10), and the combination of the forward and reverseprimers that specifically reacts with the NS5B gene of the virus, usethe following two primer pairs F1-R1, F2-R2, F1-R2 and F2-R1respectively.

The primer sets SEQ ID NO: 11-12 (PAN-FW and PAN-REV) and SEQ ID NO:13-14 (PANdeg-FW and PANdeg-REV) also specifically react with NS5B. Theset with degenerate primers SEQ ID NO: 13-14 was designed to increasethe chance of finding CTAPV variants with slightly altered RNAsequences.

The PCR-test using primer set (SEQ ID NO: 15-16) specifically reactswith the 5′ UTR of the virus and uses the two primers F3-R3.

The PCR-test using primer set (SEQ ID NO: 17-18) also specificallyreacts with the 5′ UTR of the virus and uses the two primers F4-R4

The tests, which are described in more detail in the Examples section,are standard PCR tests on cDNA. (It goes without saying that, since thevirus has an RNA genome, the viral RNA was first transcribed into cDNAin a reverse transcriptase reaction. The cDNA was used for the PCRreactions).

TABLE a Primer name Short name Sequence primer Pos. in SEQ ID NO: 19CTAPV-PAN2-F2 F2 CGGATACAGAAATACTAC 10204-10221 CTAPV-PAN2-R2 R2CCGAATGCAGCTARCAGAGG 10519-10538 CTAPV-PAN2-F1 F1 GCCATGATGGAGGAAGTG10261-10278 CTAPV-PAN2-R1 R1 GGGCAGRTTTGTGGATTCAG 10397-10416CTAPV-PAN-FW PAN-FW GAAACAGCCATGCCAAAAAATGAG 9889-9912 CTAPV-PAN-REVPAN-RV AGTGGGTTCCAGGGGTAGATCAG 10762-10784 CTAPV-PANdeg-FW PANdeg-FWGAAACAGCCATGCCMAARAATGAG 9889-9912 CTAPV-PANdeg-REV PANdeg-RVAGTGGGTTCCAGGRGTAGATYAG 10762-10784 CTAPV-PAN2-F3 F3GAGTACGGGGCAGACGTCAC 161-180 CTAPV-PAN2-R3 R3 CATCCGCCGGCACTCTATCAAGCAG318-342 CTAPV-PAN2-F4 F4 ATGCATAATGCTTTGATTGG  2-18 CTAPV-PAN2-R4 R4GTGACGTCTGCCCCGTACTC 161-180

TABLE b Anneal PCR temperature product Primer combination (° C.) size(bp) Target F1-R1 60.2 156 NS5B F1-R2 60.2 277 NS5B F2-R1 50.9 213 NS5BF2-R2 50.9 335 NS5B PAN-FW-PAN-RV 58.0 896 NS5B PANdeg-FW-PANdeg-RV 58.0896 NS5B F3-R3 50.0 182 5′-UTR F4-R4 50.0 182 5′-UTR

If a virus is characterised using the primer sets described above, thefollowing can be said: if an analysis of the PCR-product of e.g. theF1-R1 primer set reveals a PCR product of approximately 156 base pairsor if analysis of the PCR-product of e.g. the primer F2-R2 set reveals aPCR product of approximately 335 base pairs, this unequivocallydemonstrates that the analysed virus belongs to the virus according tothe invention.

Merely as an example: a PCR product of approximately 156 base pairs is aPCR product with a length of between 156+10 and 156-10 base pairs. A PCRproduct of approximately 335 base pairs is a PCR product with a lengthof between 335+10 and 335-10 base pairs.

Thus another form of this embodiment of the present invention relates toan isolated virus which is a member of the Pestiviruses, characterizedin that:

a) the virus is the causative agent of Group A-II congenital tremors inpigs and

b) the cDNA reverse-transcribed from the viral RNA genome reacts in aPCR reaction with a primer set as depicted in SEQ ID NO: 7 and 8 to givea PCR product of 156+/−10 base pairs and/or reacts in a PCR reactionwith a primer set as depicted in SEQ ID NO: 9 and 10 to give a PCRproduct of 335+/−10 base pairs and/or reacts in a PCR reaction with aprimer set as depicted in SEQ ID NO: 11 and 12 to give a PCR product of896+/−10 base pairs and/or reacts in a PCR reaction with a primer set asdepicted in SEQ ID NO: 13 and 14 to give a PCR product of 896+/−10 basepairs and/or reacts in a PCR reaction with a primer set as depicted inSEQ ID NO: 15 and 16 to give a PCR product of 182+/−10 base pairs and/orreacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 17and 18 to give a PCR product of 182+/−10 base pairs.

A preferred form of this embodiment relates to a virus according to theinvention wherein the cDNA reverse-transcribed from the viral RNA genomereacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 7and 8 to give a PCR product of 156+/−10 base pairs and reacts in a PCRreaction with a primer set as depicted in SEQ ID NO: 9 and 10 to give aPCR product of 335+/−10 base pairs and reacts in a PCR reaction with aprimer set as depicted in SEQ ID NO: 11 and 12 to give a PCR product of896+/−10 base pairs and reacts in a PCR reaction with a primer set asdepicted in SEQ ID NO: 13 and 14 to give a PCR product of 896+/−10 basepairs and reacts in a PCR reaction with a primer set as depicted in SEQID NO: 15 and 16 to give a PCR product of 182+/−10 base pairs and reactsin a PCR reaction with a primer set as depicted in SEQ ID NO: 17 and 18to give a PCR product of 182+/−10 base pairs.

A more preferred form of this embodiment relates to a virus according tothe invention wherein the virus has a viral genome comprising a geneencoding an E^(rns), a gene encoding an E2 and a gene encoding E1,wherein the nucleotide sequence of the E^(rns) gene has a level ofidentity of at least 80% to the nucleotide sequence as depicted in SEQID NO: 1 and the nucleotide sequence of the E2 gene has a level ofidentity of at least 80% to the nucleotide sequence as depicted in SEQID NO: 3 and the nucleotide sequence of the E2 gene has a level ofidentity of at least 80% to the nucleotide sequence as depicted in SEQID NO: 5 and wherein the cDNA of the viral genome reacts in a PCRreaction with a primer set as depicted in SEQ ID NO: 7 and 8 to give aPCR product of 156+/−10 base pairs and reacts in a PCR reaction with aprimer set as depicted in SEQ ID NO: 9 and 10 to give a PCR product of335+/−10 base pairs and reacts in a PCR reaction with a primer set asdepicted in SEQ ID NO: 11 and 12 to give a PCR product of 896+/−10 basepairs and reacts in a PCR reaction with a primer set as depicted in SEQID NO: 13 and 14 to give a PCR product of 896+/−10 base pairs and reactsin a PCR reaction with a primer set as depicted in SEQ ID NO: 15 and 16to give a PCR product of 182+/−10 base pairs and reacts in a PCRreaction with a primer set as depicted in SEQ ID NO: 17 and 18 to give aPCR product of 182+/−10 base pairs.

The virus according to the invention can be in a live, a live attenuatedor an inactivated form.

As indicated above, the DNA sequences of the genes encoding the E^(rns),the E2 and the E1 protein of the virus have now been characterized. Theidentification of these genes is highly useful, since they can now beused i.a. as a basis for DNA-vaccines, for use in the preparation ofsubunit vaccines on the basis of these proteins or for diagnosticpurposes, as will extensively be explained below.

Thus, another embodiment of the present invention relates to a geneencoding an E^(rns) protein characterized in that the nucleotidesequence of that gene has a level of identity of at least 80% to thenucleotide sequence of the E^(rns) gene as depicted in SEQ ID NO: 1.

A preferred form of this embodiment relates to such a gene having alevel of identity of at least 82%, more preferably 84%, 86%, 88%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%, in that orderof preference, to the nucleotide sequence of the E^(rns) gene asdepicted in SEQ ID NO: 1.

Again another embodiment of the present invention relates to a geneencoding an E2 protein characterized in that the nucleotide sequence ofthat gene has a level of identity of at least 80% to the nucleotidesequence of the E2 gene as depicted in SEQ ID NO: 3.

A preferred form of this embodiment relates to such a gene having alevel of identity of at least 82%, more preferably 84%, 86%, 88%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%, in that orderof preference, to the nucleotide sequence of the E2 gene as depicted inSEQ ID NO: 3.

And again another embodiment of the present invention relates to a geneencoding an E1 protein characterized in that the nucleotide sequence ofthat gene has a level of identity of at least 80% to the nucleotidesequence of the E1 gene as depicted in SEQ ID NO: 5.

A preferred form of this embodiment relates to such a gene having alevel of identity of at least 82%, more preferably 84%, 86%, 88%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%, in that orderof preference, to the nucleotide sequence of the E1 gene as depicted inSEQ ID NO: 5.

Still another embodiment of the present invention relates to an E^(rns)protein characterized in that this E^(rns) protein is encoded by anE^(rns) gene according to the invention.

Such E^(rns) proteins of the virus according to the invention are highlysuitable because they are i.a. suitable for use in vaccines, morespecifically in subunit vaccines, they can be used to raise antibodiesand they make diagnostic tests possible, as explained below.

A preferred form of this embodiment relates to an E^(rns) having theamino acid sequence as depicted in SEQ ID NO: 2.

Again another embodiment of the present invention relates to an E2protein, characterized in that that E2 protein is encoded by an E2 geneaccording to the invention.

Such E2's of the virus according to the invention are highly suitablebecause they are i.a. suitable for use in vaccines, more specifically insubunit vaccines, they can be used to raise antibodies and they makediagnostic tests possible, as explained below.

A preferred form of this embodiment relates to an E2 protein having theamino acid sequence as depicted in SEQ ID NO: 4.

And again another embodiment of the present invention relates to an E1protein, characterized in that that E1 protein is encoded by an E1 geneaccording to the invention.

Such E1 proteins of the virus according to the invention are highlysuitable because they are i.a. suitable for use in vaccines, morespecifically in pseudo-particles and vaccine comprising suchpseudo-particles, as explained below.

A preferred form of this embodiment relates to an E1 protein having theamino acid sequence as depicted in SEQ ID NO: 6.

It is one of the merits of the present invention that it is now for thefirst time possible to follow the course of viral infection and toanalyse the presence or absence of the novel virus in the various organsand body fluids of pigs suspected of being infected with the novel virusaccording to the invention.

It is described in the Examples section that many tissues and organsfrom pigs suffering from Group A-II congenital tremor could now betested for the presence or absence and the amount of the novel virus.

It was found that serum, plasma, PBLs, heart, small and large intestine,brain, thoracic spinal cord, lumbar spinal cord, liver, inguinal lymphnode, lung, gall bladder, bladder, kidney, tonsil and spleen isolatedfrom pigs suffering from Group A-II congenital tremor contain the novelvirus.

This helped to gain more insight in the development of the disease.

It is another merit of the present invention that it is now possible toinfect healthy pigs with the novel virus and to examine the route ofviral infection. It is described in the Examples how, with this aim,organ material from Group A-II congenital tremors-animals was isolatedand purified. This material was subsequently injected in healthypost-weaning piglets to study replication of the virus in vivo followingmethods applied by Patterson (10-20% (w/v) homogenates injected viavarious routes of administration, oral, nasal, intramuscular,subcutaneous).

It is again another merit of the present invention that it is nowpossible to infect pregnant gilts with the novel virus with the aim ofshowing that the virus is capable of causing Group A-II congenitaltremors in the piglets of these gilts. The results of these experimentsare described in the Examples.

In addition this material has been used as challenge material invaccination/challenge tests as described below.

It is also one of the merits of the present invention that, because thenovel porcine pestivirus has now been isolated, the virus and/orprotective subunits of the virus can be used as the starting materialfor vaccination purposes.

Merely as an example: the Examples section i.a. described thepreparation of vaccines comprising baculo-expressed E2 protein, theadministration of whole cell vaccines and purified E2-vaccines and asubsequent challenge with the virulent challenge material describedabove.

Thus, another embodiment of the present invention relates to vaccinesfor combating Group A-II CT in pigs, wherein such vaccines comprise animmunogenically effective amount of virus according to the invention anda pharmaceutically acceptable carrier.

Combating in this respect should be interpreted in a broad sense:combating Group A-II CT in pigs is considered to comprise vaccination inorder to prevent the signs of the disease as well as vaccination todiminish the signs of the disease as outlined above.

Examples of pharmaceutically acceptable carriers that are suitable foruse in a vaccine according to the invention are sterile water, saline,aqueous buffers such as PBS and the like. In addition a vaccineaccording to the invention may comprise other additives such asadjuvants, stabilizers, anti-oxidants and others, as described below.

A vaccine according to the invention may i.a. comprise the virusaccording to the invention in attenuated live or inactivated form.

Attenuated live virus vaccines, i.e. vaccines comprising the virusaccording to the invention in a live attenuated form, have the advantageover inactivated vaccines that they best mimic the natural way ofinfection. In addition, their replicating abilities allow vaccinationwith low amounts of viruses; their number will automatically increaseuntil it reaches the trigger level of the immune system. From thatmoment on, the immune system will be triggered and will finallyeliminate the viruses.

A live attenuated virus is a virus that has a decreased level ofvirulence when compared to virus isolated from the field. A virus havinga decreased level of virulence is considered a virus that inducesprotection against Group A-II CT or at least diminishes the symptoms ofCT, compared to the symptoms of CT caused by a wild-type pestivirusaccording to the invention.

Therefore, one preferred form of this embodiment of the inventionrelates to a vaccine comprising a virus according to the inventionwherein said virus is in a live attenuated form.

Attenuated viruses can be obtained in various ways known in the art.They can e.g. be obtained by growing a virus according to the inventionin the presence of a mutagenic agent, followed by selection of virusthat shows a decrease in progeny level and/or in replication speed. Manysuch mutagenic agents are known in the art.

Another often used method is serial in vitro passage on a susceptiblecell line. Viruses then get adapted to the cell line used for the serialpassage, so that they behave attenuated when transferred to the naturalhost again as a vaccine.

Still another way of obtaining attenuated viruses is subjecting virusesto growth under temperatures deviating from the temperature of theirnatural habitat. Selection methods for temperature sensitive mutants(Ts-mutants) are well-known in the art. Such methods comprise growingviruses, usually in the presence of a mutagen, followed by growth atboth a sub-optimal temperature and at the optimal temperature, titrationof progeny virus on cell layers and visual selection of those plaquesthat grow slower at the optimal temperature. Such small plaques compriseslow-growing and thus desired live attenuated viruses.

An alternative way to obtain a live attenuated pestivirus according tothe invention relates to the deliberate modification of the genome ofthe pestivirus. This approach has the advantage over classicalattenuation techniques as described above, that the nature of theattenuation is known. For pestiviruses, many live attenuated virusstrains of e.g. the pestiviruses Bovine Viral Diarrhea virus andClassical Swine Fever virus have been described from which e.g. the E2gene, the E^(rns) gene or the N^(pro) gene is either deleted ormodified.

Examples of live attenuated pestiviruses, more specifically the porcinepestivirus Classical Swine Fever virus (CSFV), having anN^(pro)-deletion are described i.a. in U.S. Pat. No. 7,572,455 and inMayer, D. et al⁽¹⁹⁾.

Examples of live attenuated pestiviruses, more specifically ClassicalSwine Fever virus, having both an E^(rns)-modification and anN^(pro)-deletion are described i.a. in U.S. Pat. No. 7,572,455.

Examples of live attenuated pestiviruses, more specifically ClassicalSwine Fever virus, having a modification in the E2-gene are i.a.described by Risatti, G. R. et al.⁽²²⁾ and by Risatti, G. R. et al.⁽²³⁾.

Pestiviral infections in general are a problem in many countries wherepigs, ruminants or sheep are raised. At present, different approaches todeal with pestiviral infections in general are applied in the variouscountries where pestiviruses cause economic damage. Some countries usestamping-out methods to remove the virus, whereas other countries prefera vaccination approach. The fact that these different approaches areused in parallel however causes problems. Merely as an example: e.g.porcine pestiviruses circulate in farmed pigs but also in wildlifeanimals such as wild boars, and these thus form a reservoir from whichvirus can spill into domestic animals. Animals that have been vaccinatedwith a classical vaccine cannot easily be discriminated fromfield-infected cattle, because in both cases antibodies against thevirus will be present. Thus it is largely unknown if pestiviralantibody-positive animals are antibody-positive due to infection (inwhich case they may carry the virus) or due to vaccination. As aconsequence, such animals will not be allowed to be transported tocountries that have chosen a stamping-out approach for that pestivirus.

Since the novel pestivirus causing Group A-II CT has now beenidentified, the same may apply in the future for this novel pestivirus.

This problem can be solved through the use of so-called marker or DIVAvaccines (DIVA=Differentiating Infected from Vaccinated Animals). Suchvaccines lack one or more of the immunogenic viral proteins or at leastone of the immunogenic epitopes, as a result of which marker-vaccinatedanimals will not produce antibodies against all immunogenic viralproteins/epitopes. The differences in antibody-palette betweenvaccinated and infected animals can be demonstrated by diagnostic testsdesigned for this purpose. Such tests thus allow to distinguishvaccinated from infected animals.

Since the genes encoding the E^(rns), the Npro, the E1 and the E2protein of the novel pestivirus according to the invention are nowknown, the known marker vaccine techniques as described for e.g. theporcine pestivirus CSFV can now be applied on the new virus. Examples oflive attenuated CSFV vaccines that also suitable as marker vaccines aree.g. described by Van Gennip, H. G. P. et al⁽⁷⁾, Reimann, I. et al⁽⁸⁾,Beer, M. et al⁽⁹⁾, Wehrle, F. et al⁽¹⁰⁾, by Dong, X. N. and Chen, Y.H.⁽¹¹⁾, and by de Smit, A. J. et al.⁽²⁴⁾. In most cases chimeric virusesare reported in which the E2 or E^(rns) gene was exchanged for therespective gene of a heterologous virus strain or another pestivirus.

A possible disadvantage of the use of live attenuated viruses howevermight be that inherently there is a certain level of virulence left.This is not a real disadvantage as long as the level of virulence isacceptable, i.e. as long as the vaccine at least prevents the pigs fromdying. Of course, the lower the rest virulence of the live attenuatedvaccine is, the less influence the vaccination has on weight gainduring/after vaccination.

An alternative for the use of live attenuated viruses is the use ofnon-transmissible viruses. In such viruses an essential gene is deleted,and complemented in trans in a cell line that is used to grow the virus.As a consequence, the progeny virus is a virus that, although capable ofinfecting a host cell, cannot replicate in that host cell. Such anon-transmissible virus closely mimics the natural infection and at thesame time the virus cannot spread. A vaccine comprising such anon-transmissible virus is very safe and in addition it is very suitableas a marker vaccine. Such vaccines have been described for e.g. theporcine pestivirus CSFV i.a. by Widjojoatmodjo, M. N. et al.⁽²⁵⁾, and byVan Gennip, H. G. et al.⁽²⁶⁾.

Inactivated vaccines are, in contrast to their live attenuatedcounterparts, inherently safe, because there is no rest virulence left.In spite of the fact that they usually comprise a somewhat higher doseof viruses compared to live attenuated vaccines, they may e.g. be thepreferred form of vaccine in pigs that are suffering already from otherdiseases. Pigs that are kept under sub-optimal conditions, such asincomplete nutrition or sub-optimal housing would also benefit frominactivated vaccines.

Therefore, another preferred form of this embodiment relates to avaccine comprising a virus according to the invention wherein said virusis in an inactivated form.

Such inactivated whole virus vaccines can be made for the novel porcinepestivirus according to the invention. As is the case for known porcinepestivirus vaccines, the production basically comprises the steps ofgrowing the novel porcine pestivirus on susceptible porcine cells,harvesting the virus, inactivating the virus and mixing the inactivatedvirus with a pharmaceutically acceptable carrier.

The standard way of inactivation is a classical treatment withformaldehyde. Other methods well-known in the art for inactivation areUV-radiation, gamma-radiation, treatment with binary ethylene-imine,thimerosal and the like. The skilled person knows how to apply thesemethods. Preferably the virus is inactivated with β-propiolactone,glutaraldehyde, ethylene-imine or formaldehyde. It goes without sayingthat other ways of inactivating the virus are also embodied in thepresent invention.

As indicated above, a virus according to the invention can be grown incell culture on susceptible porcine cells or cell lines.

Thus, another embodiment of the invention relates to a cell culturecomprising a pestivirus according to the present invention. An exampleof such a cell line is SK6.

Although whole inactivated porcine pestiviruses according to theinvention and non-transmissible porcine pestivirus viruses according tothen invention provide a good basis for inactivated vaccines, theirproduction may be expensive, depending i.a. upon the type of host cellsused, the substrate and the cell culture medium used.

In the specific case of pestiviruses, an attractive alternative for theuse of whole inactivated viruses or non-transmissible porcine pestivirusviruses according to the invention is the use of porcine pestivirussubunits, especially of E^(rns) and E2 protein.

The expression of such subunits, especially of E^(rns) and E2 protein isknown in the art and is extensively described for the porcine pestivirusCSFV both in baculovirus expression systems and in mammalian cells, byHulst, M. M. et al.⁽²⁷⁾, Bouma, A. et al.⁽²⁸⁾, Van Rijn, P. A. etal.⁽²⁹⁾, Moorman, R. J. M. et al.⁽³⁰⁾, Donofrio, G. et al.,⁽³¹⁾,Lutticken D. et al.⁽³²⁾, and Floegel-Niesmann et al.⁽³³⁾.

High yield expression of E^(rns) and E2 in baculovirus expressionsystems is e.g. described in EP1049788.

Furthermore, baculovirus expression systems and baculovirus expressionvectors in general have been described extensively in textbooks such asby O'Reilly at al.⁽³⁴⁾ and Murhammer⁽³⁵⁾.

Baculovirus-based expression systems are also commercially available,e.g. from Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, Calif.92008, USA.

An alternative for Baculovirus-based expression systems are yeast-basedexpression systems. Yeast expression systems are e.g. described byGellissen et al.⁽³⁶⁾.

Donofrio, G. et al.,⁽³¹⁾ describe the expression of BVDV E2 in amammalian cell line.

Ready-to-use expression systems are i.a. commercially available fromResearch Corp. Technologies, 5210 East Williams Circle, Suite 240,Tucson, Ariz. 85711-4410 USA. Yeast and insect cell expression systemsare also e.g. commercially available from Clontech Laboratories, Inc.4030 Fabian Way, Palo Alto, Calif. 94303-4607, USA.

Expression of the E^(rns) and E2 proteins in mammalian cell basedexpression systems as described by Donofrio, G. et al⁽³¹⁾ although verysuitable, would most likely be more expensive to use when compared tothe baculovirus-based expression systems.

Thus another form of this embodiment relates to a vaccine for combatingGroup A-II CT in pigs, characterized in that said vaccine comprises animmunogenically effective amount of an E^(rns) and/or E2 and/or E1protein according to the invention and a pharmaceutically acceptablecarrier.

More preferably, such subunits are in the form of so-called pestiviruspseudo-particles.

Such pseudo-particles are basically virus-like particles that comprisethe E^(rns), E1 and E2 proteins.

However they differ from the wild-type virus in that they do notcomprise the whole pestiviral genome and therefore they are not capableof replicating in the host. As a consequence, pestiviruspseudo-particles do not have to be inactivated before use in a vaccine,and therefore they have the additional advantage that they areintrinsically safe.

Pestivirus pseudo-particles can be obtained by expression of theE^(rns), E1 and E2 proteins in a suitable expression system. Examples ofpestivirus pseudo-particles and how to produce such pseudo-particles aredescribed i.a. in EP1454981 and EP1170367.

Thus again another embodiment relates to pseudo-particles characterizedin that they comprise an E^(rns) protein according to the invention, anE2 protein according to the invention and an E1 protein according to theinvention.

The amount of pseudo-particles in a vaccine and the route ofadministration would be comparable with that of inactivated whole virusparticles, since in terms of immunogenicity and similarity of the capsidthey are comparable to inactivated whole virus particles.

Usually, an amount of between 1 and 100 μg of the novel porcinepestivirus pseudo-particles would be very suitable as a vaccine dose.From a point of view of costs, a preferred amount would be in the rangeof 1-50 μg of pseudo-particles, more preferred in the range of 1-25 μg.

A vaccine according to the invention, more specifically a vaccine on thebasis of inactivated whole virus, subunits such as E^(rns) and E2protein or pseudo-particles, preferably comprises an adjuvant.Conventional adjuvants, well-known in the art are e.g. Freund's Completeand Incomplete adjuvant, vitamin E, non-ionic block polymers, muramyldipeptides, Quill A®, mineral oil e.g. Bayol® or Markol®, vegetable oil,and Carbopol® (a homopolymer), or Diluvac® Forte. The vaccine may alsocomprise a so-called “vehicle”. A vehicle is a compound to which thepolypeptide adheres, without being covalently bound to it. Often usedvehicle compounds are e.g. aluminum hydroxide, -phosphate or -oxide,silica, Kaolin, and Bentonite.

In principle it may suffice to administer a vaccine according to theinvention just once. However, especially in the case of inactivatedvaccines, be it whole virus vaccines, sub-unit vaccines orpseudo-particle vaccines, preferably also a first and possibly a secondbooster vaccination is given. A first booster would usually be given atleast two weeks after the first vaccination. A very suitable moment fora booster vaccination is between 3 and 16 weeks after the firstvaccination. A second booster, if necessary, would usually be givenbetween 4 and 50 weeks after the first booster.

An alternative to the inactivated whole virus, subunits such as E^(rns),E2 and E1 protein or pseudo-particles approach is the use of liverecombinant vector viruses that have pigs as their host animal, ascarriers of the novel porcine pestiviral E^(rns), E2 or E1 gene.

Amongst the suitable recombinant vector viruses that have pigs as theirhost animal, several vector viruses are especially suitable as carriers:Pseudorabies virus (PRV), Porcine Adeno virus (PAV), Swine Pox virus(SPV) and Classical Swine Fever virus (CSFV). In addition, vacciniavirus has been described as a suitable vector virus.

The use of such recombinant vector viruses in vaccines has theadditional advantage that the vaccinated animals become at the same timevaccinated against both the vector virus and the novel pestivirusaccording to the invention.

The use of Pseudorabies virus (PRV) as a live recombinant vector virusfor the porcine pestivirus CSFV E2 gene is described by van Zijl etal.⁽³⁸⁾ and by Peeters et al.⁽³⁹⁾ for a replication defective PRVrecombinant vector virus.

A live recombinant porcine adenovirus (PAV) vector virus as a vectorvirus for the porcine pestivirus CSFV E2 gene is described by Hammond etal.^((40,41)).

A live recombinant Swine Pox virus (SPV) vector virus as a vector virusfor the porcine pestivirus CSFV E2 gene is described by Hahn et al.⁽⁴²⁾

In addition, vaccinia virus has been described as a suitable vectorvirus by Ruemenapf et al.,⁽³⁷⁾ who describes the expression of all fourstructural proteins, and i.a. the induction of protective immunity inpigs vaccinated with vaccinia virus recombinant vectors expressing E2.

Live attenuated CSFV virus is also very suitable as live recombinantvector virus. Merely as an example; live attenuated CSFV from which theN^(pro) gene has been deleted, has been described by Mayer etal.⁽¹⁹⁾Such a live attenuated virus allows, i.a. at the site of thedeletion of the N^(pro) gene, for the insertion of the gene encoding theE^(rns) or E2 gene. Thus, such a live recombinant CSFV virus equallyforms a very suitable vector virus for the novel porcine pestiviralE^(rns) or E2 gene.

Very suitable amounts of such live recombinant vector virus would be inthe range of 10⁵ TCID₅₀ to 5×10⁹ TCID₅₀ of vector virus per vaccinedose, depending on the level of attenuation of the virus.

The expression of the novel porcine pestiviral E^(rns), E2 or E1 genecan be brought under the control of any suitable heterologous promoterthat is functional in a mammalian cell (see below). A heterologouspromoter is a promoter that is not the promoter responsible for thetranscription of the novel porcine pestiviral E^(rns), E2 or E1 gene inthe wild-type form of the novel porcine pestivirus according to theinvention.

Therefore, another embodiment of the present invention relates to a DNAfragment comprising a gene encoding the novel porcine pestiviralE^(rns), E2 or E1 gene according to the invention, characterized in thatsaid gene is under the control of a functional heterologous promoter.

A promoter that is functional in a mammalian cell is a promoter that iscapable of driving the transcription of a gene that is locateddownstream of the promoter in a mammalian cell.

Examples of suitable promoters that are functional in a mammalian cellinclude classic promoters such as the CAG promoter (Niwa, H. et al.,Gene 108: 193-199 (1991), the (human) cytomegalovirus immediate earlypromoter (Seed, B. et al., Nature 329, 840-842, 1987; Fynan, E. F. etal., PNAS 90, 11478-11482, 1993; Ulmer, J. B. et al., Science 259,1745-1748, 1993), Rous sarcoma virus LTR (RSV, Gorman, C. M. et al.,PNAS 79, 6777-6781, 1982; Fynan et al., supra; Ulmer et al., supra), theMPSV LTR (Stacey et al., J. Virology 50, 725-732, 1984), SV40 immediateearly promoter (Sprague J. et al., J. Virology 45, 773, 1983), the SV-40promoter (Berman, P. W. et al., Science, 222, 524-527, 1983), themetallothionein promoter (Brinster, R. L. et al., Nature 296, 39-42,1982), the heat shock promoter (Voellmy et al., Proc. Natl. Acad. Sci.USA, 82, 4949-53, 1985), the major late promoter of Ad2 and the β-actinpromoter (Tang et al., Nature 356, 152-154, 1992). The regulatorysequences may also include terminator and poly-adenylation sequences.Amongst the sequences that can be used are the well-known bovine growthhormone poly-adenylation sequence, the SV40 poly-adenylation sequence,the human cytomegalovirus (hCMV) terminator and poly-adenylationsequences.

Thus the present invention also relates to a live recombinant vectorvirus comprising a DNA fragment comprising a gene encoding an E^(rns)and/or E2 and/or E1 protein according to the invention under the controlof a functional promoter.

Another form of the embodiment of the present invention that relates tovaccines, relates to a vaccine for combating Group A-II CT in pigs,characterized in that said vaccine comprises a live recombinant vectorvirus comprising a DNA fragment comprising a gene encoding an E^(rns)and/or E2 and/or E1 protein according to the invention under the controlof a functional promoter and a pharmaceutically acceptable carrier.

It goes without saying that the live recombinant vector virus should beexpressing an immunogenically effective amount of the E^(rns) and/or E2and/or E1 and/or E.

An alternative for vaccination with an inactivated whole virus vaccine,a pseudo-particle vaccine or a live recombinant vector virus, is the useof DNA vaccination.

Such DNA vaccination is based upon the introduction of a DNA fragmentcarrying the gene encoding the E^(rns), E2 or E1 protein under thecontrol of a suitable promoter, into the host animal. Once the DNA istaken up by the host's cells, the gene encoding the E^(rns), E2 or E1protein is transcribed and the transcript is translated into E^(rns), E2or E1 protein in the host's cells. This closely mimics the naturalinfection process of the porcine pestivirus.

Suitable promoters are promoters that are functional in mammalian cells,as exemplified above.

A DNA fragment carrying the gene encoding the E^(rns), E2 or E1 proteinunder the control of a suitable promoter could e.g. be a plasmid. Thisplasmid may be in a circular or linear form.

An example of successful DNA vaccination of pigs is the successfulvaccination against Classical Swine Fever virus as described by Tian, D.Y. et al.⁽⁴⁵⁾, by Sun, Y. et al.⁽⁴⁶⁾, and by Sun, Y. et al.⁽⁴⁷⁾.

Other examples of successful DNA vaccination of pigs are i.a. thesuccessful vaccination against Aujeszky's disease as described in Gerdtset al.⁽⁴³⁾ They describe a DNA vaccine wherein a DNA fragment is usedthat carries glycoprotein C under the control of the major immediateearly promoter of human cytomegalovirus. Vaccination was done four timeswith two weeks intervals with an amount of 50 μg of DNA. Vaccinatedanimals developed serum antibodies that recognized the respectiveantigen in an immunoblot and that exhibited neutralizing activity.

Another example of successful DNA vaccination of pigs is given by Gorreset al.⁽⁴⁴⁾ They described successful DNA vaccination of pigs againstboth pandemic and classical swine H1N1 influenza. They vaccinated with aprime vaccination and 2 homologous boosts at 3 and 6 weeks post priming,of a DNA vaccine comprising the HA gene of influenza H1N1 under thecontrol of a functional promoter.

Since the E2 protein of the novel pestivirus according to the inventionis the most immunogenic protein, this is the preferred protein for usein DNA vaccines. Still, it may be necessary to use the methods describedabove (⁽⁴⁵⁾,⁽⁴⁶⁾,⁽⁴⁷⁾) or to rely on additional measures as describedin⁽⁹⁾ in order to enhance the immunogenicity of the DNA vaccine.

Thus, again another form of this embodiment relates to a vaccine forcombating Group A-II CT in pigs, characterized in that said vaccinecomprises a DNA fragment comprising a gene encoding an E^(rns), E2 or E1protein according to the present invention under the control of afunctional promoter, and a pharmaceutically acceptable carrier.

It goes without saying that the DNA fragment comprising a gene encodingan E^(rns), E2 or E1 protein should be expressing an immunogenicallyeffective amount of E^(rns), E2 or E1 protein.

What constitutes an “immunogenically effective amount” for a vaccineaccording to the invention that is based upon a whole porcine pestivirusaccording to the invention, a pseudo-particle according to theinvention, a live recombinant vector or a DNA vaccine according to theinvention depends on the desired effect and on the target organism.

The term “immunogenically effective amount” as used herein relates tothe amount of CTAPV, pseudo-particle, live recombinant vector or DNAvaccine that is necessary to induce an immune response in pigs to theextent that it decreases the pathological effects caused by infectionwith a wild-type Group A-II CT pestivirus, when compared to thepathological effects caused by infection with a wild-type Group A-II CTpestivirus in non-immunized pigs.

It is well within the capacity of the skilled person to determinewhether a treatment is “immunogenically effective”, for instance byadministering an experimental challenge infection to vaccinated animalsand next determining a target animal's clinical signs of disease,serological parameters or by measuring re-isolation of the pathogen,followed by comparison of these findings with those observed infield-infected pigs.

The amount of virus administered will depend on the route ofadministration, the presence of an adjuvant and the moment ofadministration. This is exemplified below and, in addition, theliterature quoted above and below relating to vaccines for otherpestivirus vaccines provides further guidance.

A preferred amount of a live vaccine comprising virus according to theinvention is expressed for instance as Tissue Culture Infectious Dose(TCID50). For instance for a live virus a dose range between 10 and 10⁹TCID50 per animal dose may advantageously be used, depending on the restvirulence of the virus.

Preferably a range between 10² and 10⁶ TCID50 is used.

Many ways of administration can be applied, all known in the art.Vaccines according to the invention are preferably administered to theanimal via injection (intramuscular or via the intraperitoneal route) orper os.

The protocol for the administration can be optimized in accordance withstandard vaccination practice. In all cases, administration through anintradermal injector (IDAL) is a preferred way of administration.

If a vaccine comprises inactivated virus or pseudo-particles accordingto the invention, the dose would also be expressed as the number ofvirus particles to be administered. The dose would usually be somewhathigher when compared to the administration of live virus particles,because live virus particles replicate to a certain extent in the targetanimal, before they are removed by the immune system. For vaccines onthe basis of inactivated virus, an amount of virus particles in therange of about 10⁴ to 10⁹ particles would usually be suitable, dependingon the adjuvant used.

If a vaccine comprises subunits, e.g. an E^(rns), E2 or E1 proteinaccording to the invention, the dose could also be expressed inmicrograms of protein. For vaccines on the basis of subunits, a suitabledose would usually be in the range between 5 and 500 micrograms ofprotein, again depending on the adjuvant used.

If a vaccine comprises a DNA fragment comprising a gene encoding anE^(rns), E2 or E1 protein, the dose would be expressed in micrograms ofDNA. For vaccines on the basis of subunits, a suitable dose wouldusually be in the range between 5 and 500 micrograms of DNA, i.a.depending on the efficiency of the expression plasmid used. In manycases an amount of between 20 and 50 micrograms of plasmid per animalwould be sufficient for an effective vaccination.

A vaccine according to the invention may take any form that is suitablefor administration in the context of pig farming, and that matches thedesired route of application and desired effect. Preparation of avaccine according to the invention is carried out by means conventionalto the person skilled in the art of making pestiviral vaccines.

Oral routes are preferred when it comes to ease of administration of thevaccine.

For oral administration the vaccine is preferably mixed with a suitablecarrier for oral administration i.e. cellulose, food or a metabolisablesubstance such as alpha-cellulose or different oils of vegetable oranimal origin.

In practice, swine are vaccinated against a number of differentpathogenic viruses or micro-organisms. Therefore it is highlyattractive, both for practical and economic reasons, to combine avaccine according to the invention for pigs with e.g. an additionalimmunogen of a virus or micro-organism pathogenic to pigs, or geneticinformation encoding an immunogen of said virus or micro-organism.

Thus, a preferred form of this embodiment relates to a vaccine accordingto the invention, wherein that vaccine comprises at least one otherpig-pathogenic microorganism or pig-pathogenic virus and/or at least oneother immunogenic component and/or genetic material encoding said otherimmunogenic component, of said pig-pathogenic microorganism orpig-pathogenic virus. An immunogen or immunogenic component is acompound that induces an immune response in an animal. It can e.g. be awhole virus or bacterium, or a protein or a sugar moiety of that virusor bacterium.

The most common pathogenic viruses and micro-organisms that arepathogenic for swine are Brachyspira hyodysenteriae, African Swine Fevervirus, Nipah virus, Porcine Circovirus, Porcine Torque Teno virus,Pseudorabies virus, Porcine influenza virus, Porcine parvovirus, Porcinerespiratory and Reproductive syndrome virus (PRRS), Porcine EpidemicDiarrhea virus (PEDV), Foot and Mouth disease virus, Transmissiblegastro-enteritis virus, Rotavirus, Escherichia coli, Erysipelorhusiopathiae, Bordetella bronchiseptica, Salmonella cholerasuis,Haemophilus parasuis, Pasteurella multocida, Streptococcus suis,Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae.

Therefore, a more preferred form of the invention relates to a vaccineaccording to the invention, wherein the virus or micro-organismpathogenic to swine is selected from the group of Brachyspirahyodysenteriae, African Swine Fever virus, Nipah virus, PorcineCircovirus, Porcine Torque Teno virus, Pseudorabies virus, Porcineinfluenza virus, Porcine parvovirus, Porcine respiratory andReproductive syndrome virus (PRRS), Porcine Epidemic Diarrhea virus(PEDV), Foot and Mouth disease virus, Transmissible gastro-enteritisvirus, Rotavirus, Escherichia coli, Erysipelo rhusiopathiae, Bordetellabronchiseptica, Salmonella cholerasuis, Haemophilus parasuis,Pasteurella multocida, Streptococcus suis, Mycoplasma hyopneumoniae andActinobacillus pleuropneumoniae.

Still another embodiment relates to a method for the preparation of avaccine according to the invention wherein the method comprises themixing of a virus according to the invention and/or an E^(rns) proteinaccording to the invention and/or an E2 protein according to theinvention and/or an E1 protein according to the invention and/or a DNAfragment according to the invention and/or a DNA fragment according tothe invention and/or a DNA fragment according to the invention and/or alive recombinant vector virus according to the invention and/or apseudo-particle according to the invention, and a pharmaceuticallyacceptable carrier.

Again another embodiment of the present invention relates to a virusaccording to the invention and/or an E^(rns) protein according to theinvention and/or an E2 protein according to the invention and/or an E1protein according to the invention and/or a DNA fragment according tothe invention and/or a DNA fragment according to the invention and/or aDNA fragment according to the invention and/or a live recombinant vectorvirus according to the invention and/or a pseudo-particle according tothe invention, for use in a vaccine for combating Group A-II CT in pigs.

As mentioned above, A-II CT is frequently found, which means that it isimportant to know if the novel pestivirus according to the invention ispresent on a farm or in a certain pig-population well before the firstclinical signs become manifest. Thus, for efficient protection againstdisease, a quick and correct detection of the presence of the novelpestivirus according to the invention is important.

Therefore it is another objective of this invention to providediagnostic tools suitable for the detection of novel pestivirusaccording to the invention.

These tools partially rely on the availability of antibodies against thevirus. Such antibodies can e.g. be used in diagnostic tests for novelpestivirus according to the invention.

Antibodies or antiserum comprising antibodies against the novelpestivirus according to the invention can quickly and easily be obtainedthrough vaccination of e.g. pigs, poultry or e.g. rabbits with the virusaccording to the invention followed, after about four weeks, bybleeding, centrifugation of the coagulated blood and decanting of thesera. Such methods are well-known in the art.

Other methods for the preparation of antibodies raised against the novelpestivirus according to the invention, which may be polyclonal,monospecific or monoclonal (or derivatives thereof) are also well-knownin the art. If polyclonal antibodies are desired, techniques forproducing and processing polyclonal sera are well-known in the art fordecades, see e.g. Mayer and Walter⁽³⁵⁾.

Monoclonal antibodies, reactive against the virus according to theinvention can be prepared by immunizing inbred mice by techniques alsolong known in the art, see e.g. Kohler and Milstein⁽³⁶⁾.

Thus, another embodiment of the present invention relates to antibodiesor antisera that are reactive with a virus according to the invention.

A diagnostic test kit based upon the detection of CTAPV may e.g.comprise a standard ELISA test. In one example of such a test the wallsof the wells of an ELISA plate are coated with antibodies directedagainst the virus. After incubation with the material to be tested,labeled antibodies reactive with the virus are added to the wells. Ifthe material to be tested would indeed comprise the novel pestivirusaccording to the invention, this virus would bind to the antibodiescoated to the wells of the ELISA. Labeled antibodies reactive with thevirus that would subsequently be added to the wells would in turn bindto the virus and a color reaction would then reveal the presence ofantigenic material of the virus.

Therefore, still another embodiment of the present invention relates todiagnostic test kits for the detection of Group A-II congenital tremorassociated porcine pestivirus, that comprise antibodies reactive with avirus according to the invention or with antigenic material thereof.Antigenic material of the virus is to be interpreted in a broad sense.It can be e.g. the virus in a disintegrated form, or viral envelopematerial comprising viral outer membrane proteins. As long as thematerial of the virus reacts with antiserum raised against the virus,the material is considered to be antigenic material.

A diagnostic test kit based upon the detection in serum of antibodiesreactive with Group A-II congenital tremor associated porcine pestivirusmay also e.g. comprise a standard ELISA test. In such a test the wallsof the wells of an ELISA plate can e.g. be coated with the virusaccording to the invention or antigenic material thereof. Afterincubation with the material to be tested, e.g. serum of an animalsuspected from being infected with the novel pestivirus according to theinvention, labeled antibodies reactive with the virus according to theinvention are added to the wells. If anti-novel pestivirus according tothe invention antibodies would be present in the tested serum, theseantibodies will bind to the viruses coated to the wells of the ELISA. Asa consequence the later added labeled antibodies reactive with the viruswould not bind and no color reaction would be found. A lack of colorreaction would thus reveal the presence of antibodies reactive with thevirus according to the invention.

Therefore, still another embodiment of the present invention relates todiagnostic test kits for the detection of antibodies reactive with GroupA-II congenital tremor associated porcine pestivirus that comprise thevirus according to the invention or antigenic material thereof.

The design of the immunoassay may vary. For example, the immunoassay maybe based upon competition or direct reaction. Furthermore, protocols mayuse solid supports or may use cellular material. The detection of theantibody-antigen complex may involve the use of labeled antibodies; thelabels may be, for example, enzymes, fluorescent-, chemoluminescent-,radio-active- or dye molecules.

Suitable methods for the detection of antibodies reactive with a virusaccording to the present invention in the sample include, in addition tothe ELISA mentioned above, immunofluorescence test (IFT) and Westernblot analysis.

An alternative but quick and easy diagnostic test for diagnosing thepresence or absence of a Group A-II congenital tremor associated porcinepestivirus is a PCR test as referred to above, comprising a PCR primerset specifically reactive with the genome of novel pestivirus accordingto the invention. Specific in this context means unique for e.g. thegenome of novel pestivirus according to the invention, i.e. not with thegenome of other pestiviruses.

It goes without saying, that more primers can be used than the primersidentified above. The present invention provides for the first time theunique sequence of the genome of the novel pestivirus according to theinvention. This allows the skilled person to select without anyadditional efforts, other selective primers. By simple computer-analysisof the genome of novel pestivirus according to the invention genesequence provided by the present invention with the, known, genome ofother pestiviruses, the skilled person is able to develop other specificPCR-primers for diagnostic tests for the detection of a novel pestivirusaccording to the invention and/or for distinguishing between an novelpestivirus according to the invention and other viral (porcine)pathogens.

PCR-primers that specifically react with the genome of novel pestivirusaccording to the invention are understood to be those primers that reactonly with the genome of novel pestivirus according to the invention andnot with the genome of another (porcine) pathogenic virus, or group of(porcine) pathogenic viruses.

Thus, another embodiment relates to a diagnostic test kit for thedetection of Group A-II congenital tremor associated porcine pestivirus,characterised in that said test kit comprises a PCR primer set that isspecifically reactive with the genome of the novel pestivirus accordingto the invention.

A preferred form of this embodiment relates to a diagnostic test kit forthe detection of Group A-II congenital tremor associated porcinepestivirus, wherein said test comprises the primer set as depicted inSEQ ID NO: 15-16.

A special form of a diagnostic test is provided by the qRT-PCR testdescribed in more detail in Example 10. This test is very suitable forthe quantification of the amount of virus present in various samplessuch as serum samples, sperm samples and tissue samples. Such testsallow, in addition to the detection of viral RNA, for a quick andreliable quantification of the number of RNA copies present in suchsamples.

In Example 10, it is described how RNA was isolated and subjected toRT-reactions, whereafter oligonucleotide primers were used to amplifythe 5′ UTR genome of the CTAPV genome. This part of the viral genome waschosen based on conserved nucleotide sequence between CTAPV variants 1-9(based on alignment of the nucleotide sequences). The primer sequencesused in Example 10 were as follows: CTAPV-PAN2-F3-B:CGTGCCCAAAGAGAAATCGG (SEQ ID NO: 35) and CTAPV-PAN2-R3-B (SEQ ID NO:36): CCGGCACTCTATCAAGCAGT.

The skilled person would however realise that any part of the viralgenome that shows a conserved nucleotide sequence between CTAPV variantscan be used for the selection of suitable primers.

Example 10 shows how the qRT-PCR reaction according to the invention wassuccessfully used for the detection of viral RNA in e.g. the sperm ofboars.

In Example 11 it is shown, using this diagnostic technique, thatCTAPV-free gilts can become infected with CTAPV through the sperm ofCTAPV-infected boars.

LITERATURE

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LEGEND TO THE FIGURES

FIG. 1: Schematic overview of primers designed in the RNA polymerasegene (NS5B) of CTAPV, and PCR products.

FIG. 2: Formalin fixed and hematoxyline-eosine stained 400×magnifications of the most distinct abnormalities in brain and spinalcord tissue. (A) Cross section of the cerebellum that showsvacuolisation of Purkinje cells (the layer of large cells between thegranular layer and the white matter. White arrows show examples ofvacuolization in some of the Purkinje cells. (B) Vacuolisation of thewhite matter, indicative for demyelination. Some examples ofdemyelination of axons in the spinal cord are indicated by white arrows.(C) Accumulation of microglia (stained dark purple) forming a microglialnodule around a degenerating neuron (neuronophagia) in the cerebrum. Theneuron is indicated by the white arrow. (D) Perivascular cuffing in thethoracic spinal cord. Eosinophilic granulocytes are surrounding a bloodvessel which is indicated by the arrows.

FIG. 3: Phylogenetic tree of CTAPV 1 and other previously identifiedpestiviruses of which the nucleotide sequence was deposited in Genbank(accession numbers indicated in the Figure). The amino acid sequences ofthe polyprotein were used for the nearest neighbor method. The bar inthe left corner presents the average number of nucleotidesubstitution/site.

FIG. 4: Phylogenetic analysis of CTAPV variants. The amino acidsequences are used for the nearest neighbor method. The bar in the leftcorner presents the average number of nucleotide substitution/site.Analysis based on the first 5000 nucleotides of the genome. CTAPV type 7not included. CTAPV 5 is identical to CTAPV 8.

FIG. 5: Amino acid sequence comparison of Erns-E1-E2 region of CTAPV 1and 1B. The E2 protein sequence is in Italics. The Ems protein isunderlined with a thick line, the E1 protein sequence is underlined witha thin line.

The subject sequence in FIG. 5 that is the thick underlined matches SEQID NO: 2. The subject sequence in FIG. 5 in italics matches SEQ ID NO:4. The subject sequence in FIG. 5 that is thin underlined matches SEQ IDNO: 6.

Sequence ID NO: 21 is a partial genome sequence of CTAPV 1B (also calledM1B in the application) in which includes the nucleotide sequences ofEms, E1 and E2. The Query amino acid sequence of FIG. 5 can betranslated from this nucleotide sequence (nucleotides 1247-3190).

FIG. 6: Amino acid sequence comparison of Erns-E1-E2 region of CTAPV 1Band 8. The E2 protein sequence is in Italics. The Ems protein isunderlined with a thick line, the E1 protein sequence is underlined witha thin line.

Sequence ID NO: 21 is a partial genome sequence of CTAPV_1B (also calledM1B in the application) in which includes the nucleotide sequences ofEms, E1 and E2. The Query amino acid sequence of FIG. 6 can betranslated from this nucleotide sequence (nucleotides 1247-3190).

Sequence ID NO:28 is a partial genome sequence of CTAPV_8 (also calledM8 in the application) in which includes the nucleotide sequences ofEms, E1 and E2. The Subject amino acid sequence of FIG. 6 can betranslated from this nucleotide sequence (nucleotides 1170-3113).

FIG. 7: Antibodies generated in rabbits specifically recognize the CTAPVE2 protein expressed in the baculovirus/SF9 expression system. Markerbands correspond (from bottom to top) to 5, 10, 20, 25, 37, 50, 75, 100,150 and 250 kDa.

FIG. 8: Indication of the location of the Ems protein coding region(thick underlined), the E1 protein coding region (thin underlined) andthe E2 protein coding region (in Italic). Sequence starts at nt 1259 ofthe reference genome. FIG. 8 is the Erns-E1-E2 nucleotide sequence andmatches Seq ID: 19. The sequence in FIG. 8 that is thick underlinedmatches SEQ ID NO: 1. The sequence in FIG. 8 in italics matches SEQ IDNO: 3. The sequence in FIG. 8 that is thin underlined matches SEQ ID NO:5.

FIG. 9: RT-qPCR data of the standard line samples and the negativecontrol sample. FIG. 9 A shows a diagram with Ct values with cyclesplotted against RFU, FIG. 9 B shows the standard curve; Ct valuesplotted against log-transformed concentrations of serial ten-fold (log)dilutions of the target nucleic acid and FIG. 9 C shows the derivativemelting curve in Real Time.

EXAMPLES Example 1

Discovery of New Virus, CTAPV 1, on a Pig Farm in the Netherlands.

On a pig farm located in the Netherlands, an outbreak of congenitaltremor type A-II was diagnosed in early 2012. Piglets born from gilt,first parity animals, were primarily affected but also higher paritysows were occasionally affected. Diagnosis was based on clinicalobservations and subsequent exclusion of congenital tremor types A-I,A-III, A-IV and A-V as the possible cause for disease. Clinically,affected piglets showed tremor in different grades, due to excessivemuscle contractions during activity. The symptoms diminished whensleeping. Piglet loss was a secondary effect caused by the inability ofaffected animals to feed themselves, especially during the first weekafter birth. Histologically, the brain and the spinal cord werecharacterized by hypomyelinization. As further described below, not allaffected pigs survived. In those that survived, the tremor diminishedand finally disappeared as pigs grew older.

Based on the outbreak information, an infectious origin of the diseasewas suspected. In the first 20 weeks of the year 2012, a total of 48μlitters with symptoms of congenital tremor were born from gilts, out of231 litters born from gilts in total. This equals 21% of all littersborn from gilts. At the peak of infection, 8 weeks after the initialoutbreak, 85% of the gilt litters showed piglets with congenital tremortype A-II. The percentage piglet loss (piglet death) till weaning was26% in affected litters, compared to 11% in non-affected litters. Inaffected litters, 60% of piglet death was attributable to congenitaltremor. The total number of piglets born per litter was not affected.Congenital tremor affected both sexes, and prevalence within the littervaried between <10%-100%.

Prior to the outbreak in 2012, congenital tremor was observed in a fewlitters in November 2009 and December 2010.

Problems with outbreaks of congenital tremor have continued on this farmsince 2012, and affected piglets were obtained in 2013 and 2014 (seebelow). However, the incidence rate decreased.

Blood plasma samples were obtained in March 2012 (6 samples, all pigletswith symptoms of CT type A-II) and April 2012 (5 samples, all pigletswith symptoms of CT type A-II). The new virus CTAPV 1 was detected in11/11 samples.

More blood plasma samples were obtained from the same farm in July 2012.A total of 16 serum samples from piglets born from 2 sows and 1 giltwere analyzed. None of these piglets showed congenital tremor. CTAPV wasfound in 1/16 samples.

A new outbreak of the disease was diagnosed in January 2013. Fournewborn pre-colostral piglets were obtained for necropsy, all showed CTtype A-II. This virus was named CTAPV 1A because it originated from thesame farm, but significant time had elapsed between the originaloutbreak and the occurrence of new clinical problems. The new virusCTAPV 1A was detected in 4/4 piglets.

A new outbreak of the disease was diagnosed in March 2013. Three newbornpre-colostral piglets were obtained for necropsy, all showed CT typeA-II. This virus was named CTAPV 1B. The new virus CTAPV 1 was detectedin 3/3 samples.

A new outbreak of the disease was diagnosed in January 2014. Fournewborn pre-colostral piglets were obtained (rectal swabs), all showedCT type A-II. This virus was named CTAPV 1C. The new virus CTAPV 1 wasdetected in 4/4 samples. Necropsy on an additional 3 piglets wasperformed in February 2014, again all 3 piglets showed CT type A-II, andCTAPV was detected in 3/3 samples.

Post mortem examination was performed on piglets from outbreaks inJanuary 2013, March 2013 and February 2014. Brains and spinal cordshowed signs of demyelinization (see Example 2).

Seven piglets (6 pre-partus, last week of gestation; 1 newborn) from afarm with no history of congenital tremor type A-II were used asnegative control for PCR and for post mortem examination. All plasmasamples were negative for CTAPV virus, and no histological abnormalitieswere observed in these piglets.

Collection of Serum and Feces Samples

Feces and serum samples were obtained at farms in the Netherlands thathave problems with CT type A-II in newborn pigs. Blood was collected ina tube (type: Vacuolette 8 ml Sep Clot Activator ref: 455071) and serumwas isolated by centrifuging 20 minutes at 3000×g at 4° C. Feces werecollected using a dry cotton-swab and put in a sterile tube containing 2ml Phosphate-buffered saline solution (PBS). Then cotton swabs withfeces were stirred strongly and discarded. Both serum and feces sampleswere stored at −70° C. until analysis.

Viral RNA Isolation with Optional DNAse Treatment

For viral RNA isolation, the QIAamp Viral RNA mini Kit (Qiagen) was usedin combination with RNase free DNase kit (Qiagen).

In short, 1% solution of carrier-RNA/AVE in AVL buffer was prepared. 560μl carrier-RNA/AVE in AVL was mixed with 140 μl sample and incubated 10minutes at room temperature. Then 560 μl ethanol (>99%) was added andsamples were transferred to a QIAamp mini spin column. Columns werecentrifuged for 1 minute at 6000×g. Columns were washed by adding 250 μlAW1 and spinning the columns 30 seconds at 6000×g. DNase-mix wasprepared by mixing 10 μL DNase with 70 μl RDD buffer per sample. 80 μlDNase-mix was incubated on the membrane during 15 minutes at roomtemperature. Washing was continued by putting 250 μl AW1 on the columnand spinning it 30 seconds at 6000×g, followed by adding 500 μl AW2 tothe columns and centrifuging 3 minutes at 13000×g. Collection tubes werereplaced and columns were centrifuged for another minute. Spin columnswere transferred into a 1.5 ml Eppendorf tube, where 65 μl AVE bufferwas added on membranes and centrifuged 1 minute at 6000×g. The RNAsamples were preceded to the Reverse Transcriptase-reaction immediately.

Reverse Transcriptase-Reaction

RNA was transcribed into cDNA using SuperScript® III First-StrandSynthesis System for RT-PCR (Invitrogen). The manufacturer's protocolwas followed with some minor modifications. In summary, 1 μl randomhexamers and 1 μl 10 mM dNTPs were mixed with 8 μl RNA. This was firstincubated 5 minutes at 65° C., then chilled on ice. Then 10 μl cDNAsynthesis mix, consisting of 2 μl 10×RT buffer, 4 μl MgCL₂, 2 μl DTT, 1μl RNaseOUT and 1 μl Superscript®III RT, was added to the samples. Thesamples were first incubated 10 minutes at 25° C., then 50 minutes at50° C., followed by 5 minutes at 85° C. and finally chilled on ice. 1 μlRNase H was added to the samples and this was incubated 20 minutes at37° C. The obtained cDNA samples were stored at −20° C. until use.

PCR

A. Primer combination CTAPV-PAN2-F1R1, -F2R1, -F1R2, -F2R2, Table 1,2

Each PCR reaction contained 27 μl WFI, 1 μl Super Taq Plus 5 μl 10×Super Taq PCR buffer, 5 μl dNTPs, 5 μl forward primer and 5 μl reverseprimer. Overview of used primers is depicted in Table 1. The PCR programused to detect CTAPV consisted of a 4 minute initialization-phase, at95° C. This was followed by 35 cycles of sequentially denaturation for30 seconds at 95° C., annealing for 30 seconds at the appropriateannealing temperature for the primer pair (see Table 1) and extensionfor 30 seconds at 72° C. A final extension at 72° C. was maintained for10 minutes. All PCR products were analyzed with 1.5% agarose-gelelectrophoresis. See FIG. 1.

B. Primer combination CTAPV-PAN-FW-RV, PANdeg-FW-PANdeg-REV, Table 1,2

Each PCR reaction contained 27 μl WFI, 1 μl Super Taq Plus 5 μl 10×Super Taq PCR buffer, 5 μl dNTPs, 5 μl forward primer and 5 μl reverseprimer. Overview of used primers is depicted in Table 2. The PCR programused to detect CTAPV consisted of a 4 minute initialization-phase, at95° C. This was followed by 40 cycles of sequentially denaturation for30 seconds at 95° C., annealing for 30 seconds at the appropriateannealing temperature for the primer pair (see Table 2) and extensionfor 60 seconds at 72° C. A final extension at 72° C. was maintained for10 minutes. All PCR products were analyzed with 1.5% agarose-gelelectrophoresis.

C. Primer combination CTAPV-PAN2-F3R3, -F4R4, Table 1,2

Each PCR reaction contained 27 μl WFI, 1 μl Super Taq Plus 5 μl 10×Super Taq PCR buffer, 5 μl dNTPs, 5 μl forward primer and 5 μl reverseprimer. Overview of used primers is depicted in Table 1. The PCR programused to detect CTAPV consisted of a 4 minute initialization-phase, at95° C. This was followed by 35 cycles of sequentially denaturation for30 seconds at 95° C., annealing for 30 seconds at the appropriateannealing temperature for the primer pair (see Table 1) and extensionfor 30 seconds at 72° C. A final extension at 72° C. was maintained for10 minutes. All PCR products were analyzed with 1.5% agarose-gelelectrophoresis.

TABLE 1 Overview of Primers Primer name Short name DNA SequenceSEQ ID NO CTAPV-PAN2-F2 F2 5′-CGGATACAGAAATACTAC-3′ SEQ ID NO: 9CTAPV-PAN2-R2 R2 5′-CCGAATGCAGCTARCAGAGG-3′ SEQ ID NO: 10 CTAPV-PAN2-F1F1 5′-GCCATGATGGAGGAAGTG-3′ SEQ ID NO: 7 CTAPV-PAN2-R1 R15′-GGGCAGRTTTGTGGATTCAG-3′ SEQ ID NO: 8 CTAPV-PAN-FW PAN-FW5′-GAAACAGCCATGCCAAAAAATGAG-3′ SEQ ID NO: 11 CTAPV-PAN-REV PAN-RV5′-AGTGGGTTCCAGGGGTAGATCAG-3′ SEQ ID NO: 12 CTAPV-PANdeg-FW PANdeg-FW5′-GAAACAGCCATGCCMAARAATGAG-3′ SEQ ID NO: 13 CTAPV-PANdeg-REV PANdeg-RV5′-AGTGGGTTCCAGGRGTAGATYAG-3′ SEQ ID NO: 14 CTAPV-PAN2-F3 F35′-GAGTACGGGGCAGACGTCAC-3′ SEQ ID NO: 15 CTAPV-PAN2-R3 R35′-CATCCGCCGGCACTCTATCAAGCAG-3′ SEQ ID NO: 16 CTAPV-PAN2-F4 F45′-ATGCATAATGCTTTGATTGG-3′ SEQ ID NO: 17 CTAPV-PAN2-R4 R45′-GTGACGTCTGCCCCGTACTC-3′ SEQ ID NO: 18

TABLE 2 Overview of primer combinations used, and characteristics oftargets Anneal PCR temperature product Primer combination (° C.) size(bp) Target F1-R1 60.2 156 NS5B F1-R2 60.2 277 NS5B F2-R1 50.9 213 NS5BF2-R2 50.9 335 NS5B PAN-FW-PAN-RV 58.0 896 NS5B PANdeg-FW-PANdeg-RV 58.0896 NS5B F3-R3 50.0 182 5′-UTR F4-R4 50.0 182 5′-UTRD. SYBR Green Quantitative PCRStandard Line for Quantification of qPCR Results

To obtain a standard for qPCR, a 155 bp PCR product of the CTAPVsequence containing the qPCR target sequence was cloned into a TOPO4plasmid vector (Life Technologies) according to the manufacturer'sinstructions. The 155 bp CTAPV PCR product for cloning was obtained byperforming a PCR with CTAPV-PAN2-F1 and CTAPV-PAN2-R1 primers, see Table3. Subsequently, the PCR-product was electrophoresed on a 1.5%agarose-gel. The 155 bp band was cut out and DNA was extracted from theagarose-gel prior to cloning in the TOPO4 vector.

The TOPO TA Cloning Kit (Invitrogen) was used to ligate the PCR productinto a pCR 4-TOPO4 vector and to transform this into One Shot TOP10Chemically Competent E. Coli. In summary, 4 μl of DNA was mixed with 1μl salt solution and 1 μl of TOPO vector. This ligation was incubatedfor 5 minutes at room temperature and then placed on ice. 2 μl ligationmix was added to One Shot® TOP10 Chemically Competent E. Coli. After 30minutes incubation on ice, the mixture was heat shocked in a 42° C.water bath during 30 seconds and placed back on ice. Now 250 μl warm SOCmedium was added and the mixture was incubated 1 hour at 37° C. in ashaking incubator, after which 100 μl mixture was spread out over anagar-LB+100 μg/ml ampicillin plate. The plate was incubated overnight ina 37° C. incubator.

Correctly cloned colonies were identified using colony-PCR using M13Primers (see Table 3 below; (SEQ ID NO: 30 and 31)) in standard PCRassays, followed by gel electrophoresis. The correct colonies were grownin LBACF medium (MSD AH Media Production lot. No. 318781; Luria-Bertanimedium, animal component free) with ampicillin, from which plasmid DNAwas isolated using a QIAGEN® Plasmid Midi kit (Qiagen) according tomanufacturer's protocol. To check for mutations, the plasmid DNA wassequenced using M13 primers.

TABLE 3 Overview of primer combinations used for qPCR analysis AnnealingPrimer name Primer DNA sequence SEQ ID NO Temperature CTAPV-PAN2-F15′-GCCATGATGGAGGAAGTG-3′ SEQ ID NO: 7 60.0° C. CTAPV-PAN2-R15′-GGGCAGRTTTGTGGATTCAG-3′ SEQ ID NO: 8 60.0° C. M13 Fw5′-GTAAAACGACGGCCAG-3′ SEQ ID NO: 30 55.0° C. M13 Rv5′-CAGGAAACAGCTATGAC-3′ SEQ ID NO: 31 55.0° C.

Standard dilutions of the target sequence were calculated by measuringplasmid DNA concentrations of the vector. The formula for calculatingplasmid copies/μl is depicted below (Formula 1). The DNA concentration(ng/μl) was measured using spectrophotometry. A, G, T and C are countsof the homonymous nucleotides in the plasmid. 6.02*10²³ is the number ofAvogadro. The multiplication by 2 converts ssDNA concentration intodsDNA concentration, and the multiplication by 10⁹ converts gram intonanogram. For qPCR reactions, eight dilutions were made containing10⁸-10¹ copies/2 μl.

$\left. {{{Formula}{\mspace{11mu}\;}1\text{:}\mspace{14mu}{Formula}\mspace{14mu}{for}\mspace{14mu}{calculation}\mspace{14mu}{of}\mspace{14mu}{plasmid}\mspace{14mu}{copies}\text{/}{µl}}{{{Plasmid}\mspace{14mu}{copies}\text{/}{µl}} = {{DNA}\mspace{14mu}{concentration}\;{\left( {{ng}\text{/}{µl}} \right)/\left( {\left( \frac{\left( {{A*328,24} + {G*344,24} + {T*303,22} + {C*304,16}} \right)}{\left( {6,02*{10\bigwedge 23}} \right)} \right)* 2*{10\bigwedge 9}} \right)}}}} \right).$qPCR

A SYBR green based qPCR was developed. Each reaction contained 10 μlKAPA SYBR Fast qPCR master mix, 0.4 μl 10M forward primer, 0.4 μl 10Mreverse primer, 7.2 μl WFI and 2 μl template. Primers CTAPV-PAN-F1 andCTAPV-PAN-R1 were used (See Table 4). The following program was used: 3minutes at 95° C., followed by 39 cycles of sequentially 10 seconds at95° C., 10 seconds at 60° C. and plate read in a Biorad CFX system.Results were analyzed using Biorad CFX software. Results were comparedwith a standard line as described above; a 10-fold dilution series ofthe 155 bp CTAPV product, cloned into a TOPO4 plasmid. A melting curveanalysis between 65° C.→95° C.; per 0.5° C. 0.05 seconds was included inthe qPCR program.

Specificity of the qPCR reaction was validated by gel electrophoresis ofthe amplified PCR product. The calibration curve slope and y-interceptwere calculated by the CFX software. The r² was >0.99. The PCRefficiency calculated from the slope was between 95-105%.

TABLE 4 qPCR reaction mix volume User solution (μl)/reaction KAPA SYBRFast qPCR mastermix 2× 10 CTAPV-PAN2-F1 10 μM 0.4 CTAPV-PAN2-R1 10 μM0.4 WFI n.a. 7.2 Template (cDNA) n.a. 2Nucleotide Sequencing

Sanger sequencing was performed according to methods described inliterature. Sequences were analyzed using Sequencer 5.0 and CloneManager 9.

Phylogenetic Analysis

Phylogenetic analysis was performed to categorize CTAPV 1 as apestivirus.

The amino acid sequences of the entire gene of the novel virus were usedto make phylogenetic trees based on the Neighbor-Joining MaximumLikelyhood method, the Poisson correction model and bootstrap analysis(500 replicates).

These trees were made using the program MEGA, version 5, using standardsettings. (MEGA5: Molecular Evolutionary Genetics Analysis Using MaximumLikelihood, Evolutionary Distance, and Maximum Parsimony Methods.Koichiro Tamura, Daniel Peterson, Nicholas Peterson, Glen Stecher,Masatoshi Nei and Sudhir Kumar. Mol. Biol. Evol. 28(10): 2731-2739. 2011doi:10.1093/molbev/msr121 Advance Access publication May 4, 2011).

Example 2

Virus CTAPV can be Found in Organs and PBLs; Histology Indicative forDemyelination in Brain and Spinal Cord

PCR analysis of the following organs of the necropsied pre-colostralnew-born piglets (CTAPV 1A/1B, 2013) with congenital tremor type A-IIindicated presence of CTAPV virus.

CTAPV could be detected in blood, serum, plasma, and PBLs (peripheralblood leukocytes), heart, small intestine, large intestine, brain,thoracic spinal cord, lumbar spinal cord, liver, inguinal lymph node,lung, gall bladder, bladder, kidney, tonsil and spleen. Highestquantities were detected in serum and tonsils.

The same organs were samples from pre-partus (last week of gestation)control piglets from a farm with no history of CT type A-II. All organswere negative in the PCR.

Brains and spinal cords of control and CTAPV-infected piglets werenecropsied, formalin fixed and hematoxyline-eosine stained. Histologicalexamination revealed indications for demyelination exclusively inCTAPV-infected piglets (FIG. 2 A-D).

CTAPV Variants from Farms at Different Geographical Locations.

CTAPV variants 2-9 were obtained from pig farms in the Netherlands fromoutbreaks in 2013 and onwards.

Table 5 shows the number of piglets tested on each farm, and the numberof CTAPV PCR positive piglets (serum/rectal swabs).

TABLE 5 Overview of CTAPV variants from different farms in TheNetherlands. Results of PCR analysis of CTAPV in serum and/or rectalsamples. CTAPV pos. CTAPV neg. CTAPV pos. with with without Total numberVariant Farm symptoms symptoms symptoms of samples Date CTAPV 1 1 6 0 06 15-mrt-2012 CTAPV 1 1 5 0 0 5 5-apr-2012 CTAPV 1 1 0 0 1 1520-jul-2012 CTAPV 1A 1 4 0 0 4 28-jan-2013 CTAPV 1B 1 3 0 0 3 5-mrt-2013CTAPV 1C 1 4 0 0 4 31-jan-2014 CTAPV 1C 1 3 0 0 3 12-feb-2014 CTAPV 2 28 0 0 8 14-aug-2013 CTAPV 3 3 8 0 0 8 11-okt-2013 CTAPV 4 3 0 0 4 811-okt-2013 CTAPV 5 4 5 0 0 5 31-mei-2013 CTAPV 6 5 10 0 0 10 4-dec-2013CTAPV 7 6 15 0 0 15 8-jan-2014 CTAPV 7 6 4 0 0 4 24-jan-2014 CTAPV 8 7 40 0 4 6-mrt-2014 CTAPV 9 8 4 0 0 4 12-feb-2014 NEG. CONT. 9 0 0 0 15-mrt-2013 NEG. CONT. 9 0 0 0 6 18-dec-2014 TOTAL 83 0 5 113

The disease association is 100% for piglets showing CT type A-II. CTAPVvirus was detected in all piglets with congenital tremor type II, andnot in control samples taken on a farm with no history of CT type A-II.

CTAPV 1 was found in one piglet that did not show congenital tremor.This piglet originated from Farm 1, a farm with history of CT type A-II.

CTAPV 4 was found in piglets that did not show congenital tremor. CTAPV4 was found at the same farm where CTAPV 3 was found (Farm 3). Thus,CTAPV 4 was present on a farm with history of CT type A-II.

A total of 12 variants from 8 geographical different locations werefound.

-   -   Variants CTAPV 1, 1A, 1B, 1C originate from the same farm at        different points in time.    -   Variants CTAPV 3 and 4 originate from the same farm    -   Although found at different geographical locations, Variants        CTAPV 5 and 8 are identical at the nucleotide level

Table 6 shows reactivity of primer pairs.

TABLE 6 Reactivity of primer pairs. PAN-FW - PANdeg-FW - Variant F1R1F1R2 F2R1 F2R2 F3R3 F4R4 PAN-RV PANdeg-RV CTAPV 1 + + + + + + + + CTAPV1A + + + + + + + + CTAPV 1B + + + + + + + − CTAPV 1C + na na na + + − −CTAPV2 + + + + + na + na CTAPV 3 − − + + + na na na CTAPV 4 na na nana + na na na CTAPV 5 + + + + + na + na CTAPV 6 + + + + + na na na CTAPV7 + na na + + + + + CTAPV 8 + na na + + + + + CTAPV 9 na na na na + nana na

All variants can be detected using PCR primer pair F3R3

All variants can be detected using one of the PCR primer combinationsF1R1, F1R2, F2R1, F2R2, however, Variant CTAPV 9 was not tested.

Genome Sequencing

The complete genome sequence of CTAPV 1 was obtained by Sangersequencing.

Of other variants, CTAPV 1A, 1B, 1C, 2, 3, 4, 6, 8 and 9, the first 5000bp including the coding sequences for E^(rns), E1 and E2 were obtained.

Only a limited nucleotide sequence of 1073 nt is available for M7

Based on genome sequencing, it was concluded that CTAPV 5=CTAPV 8

Example 3

Phylogenetic Analysis of CTAPV and CTAPV Variants

The phylogenetic tree of the CTAPV 1 and other known pestiviruses ispresented in FIG. 3. The percentage bootstrap support is specified atthe nodes. Distance bars indicate the number of nucleotide substitutionsper site.

The phylogenetic tree of 10 of the CTAPV variants described in thispatent application is presented in FIG. 4. Only variants CTAPV 1, 1A,1B, 1C, 2, 3, 4, 6, 8 and 9 were included in this analysis. Thenucleotide sequence 1-5000 bp were included in this analysis, whichincludes the coding sequences for E^(rns), E1 and E2.

CTAPV 7 was not included because only 1073 nt are available for M7.

CTAPV 5 is not included, because CTAPV 5=CTAPV 8

Example 4

Analysis of the Predicted E2 Protein/Nucleotide Sequence Shows thatCTAPV 1B E2 Protein=CTAPV 1 E2 Protein. CTAPV 8 Protein Shows 14 AminoAcid Substitutions Compared to CTAPV 1.

Necropsied organs that could serve as starting material for infectionexperiments were available for CTAPV 1B, but not for CTAPV 1. Weanalyzed the nucleotide and amino acid sequence of the E^(rns)-E1-E2genes/proteins of CTAPV 1 and 1B. The amino acid sequence is 100%identical (FIG. 5). The E2 protein sequence is in Italic. The E^(rns)protein is underlined with a thick line, the E1 protein sequence isunderlined with a thin line.

Necropsied organs that could serve as starting material for infectionexperiments were also available for CTAPV 8. We analyzed the nucleotideand amino acid sequence of the E^(rns)-E1-2 genes/proteins of CTAPV 1Band 8 (amino acid comparison in FIG. 6). The amino acid sequence is 95%identical. The E2 protein sequence is in Italic. The E^(rns) protein isunderlined with a thick line, the E1 protein sequence is underlined witha thin line. CTAPV 8 has 14 amino acid substitutions (93.3% identity)compared to CTAPV 1B, of which 9 are positives (positives 97.6%).

Example 5

Preparation of Challenge Material

Challenge material was obtained from necropsied organs (field material)of piglets affected by CTAPV 1B (2013) and CTAPV 8 (2014). Necropsiedorgans were stored at −70° C. until use.

CTAPV 1B

Brains of 3 piglets of the affected litter were pooled prior tohomogenization.

Spinal cord of 3 piglets of the affected litter were pooled prior tohomogenization

Spleens of 3 piglets of the affected litter were pooled prior tohomogenization

Tonsils of 3 piglets of the affected litter were pooled prior tohomogenization

CTAPV 8

Brains of 4 piglets of the affected litter were pooled prior tohomogenization

Spinal cord of 4 piglets of the affected litter were pooled prior tohomogenization

Spleens of 4 piglets of the affected litter were pooled prior tohomogenization

Tonsils of 4 piglets of the affected litter were pooled prior tohomogenization

Pooled tissues were weighted after thawing. Subsequently, 9 timestissue-weight PBS (CTAPV 1B) or M6B8 medium with 1 μM HEPES (SigmaH3375-250G, CTAPV 8) was added to the tissue material. The tissue washomogenized using a blender, followed by shaking with small glass beadsfor 5 minutes. During homogenizing organ-pulp was kept on ice. Theorgan-pulp was centrifuged 1 hour at 3200×g. Supernatant was firstpassed over a 0.45 μm filter, and subsequently over a 0.22 μm filter.The filtered homogenate was stored at −70° C. until use.

Example 6

Infection Experiment in Weaner Aged Piglets to Obtain InfectiousMaterial:

Challenge experiments with CTAPV 1B and CTAPV 8 organ homogenatesoriginating from field isolates were conducted in 4 to 8 week oldweaning-aged SPF/high health piglets of a commercial finisher pig breed.

At the time of placing in the test facility, CPDA (citrate phosphatedextrose adenine) blood samples, rectal swabs, oropharynx swabs andnasal swabs were obtained from the animals. Animals were housed in twoseparate experiment rooms: group A 8 animals and group B 8 animals.There was no physical contact or indirect contact via animal caretakersbetween the rooms.

In group A, six pigs were inoculated with CTAPV 1B homogenates via theintramuscular (IM), subcutaneous (SC), intranasal (N) and oral (OR)routes.

Two pigs received inoculum from mixed spleen+spinal cord+brainhomogenate

Two pigs received inoculum from mixed spleen+tonsil+brain homogenate

Two pigs received inoculum from mixed brain+spinal cord homogenate

Two pigs served as contact sentinels

IM, SC and N volumes were 1.0 ml per dose, left and right. OR volume was4 ml. Nasal dose was sprayed. Challenge doses are given in Table 7.

After inoculation, all pigs were observed daily for clinical signs, butthe animals remained asymptomatic during the course of the experiment.

CPDA-blood, nose swabs, oropharynx swabs and rectal swabs were taken onday 0, day 3, day 7, day 10 and day 14 after inoculation to monitorinfection and excretion of CTAPV 1B via qPCR analysis. Plasma wasobtained from CPDA blood using the Leucosep® kit (Greiner Mat. no. 163288). The results of qPCR analysis on plasma samples are presented inTable 7.

All inoculated animals showed a positive CTAPV qPCR result in bloodplasma at day 10. Based on excretion of virus, animals were sacrificedat different time points to obtain fresh infectious material forsubsequent in vitro and in vivo studies.

At the time of necropsy, brain, spinal cord, spleen, tonsils, and bloodwere taken from the animals.

TABLE 7 Challenge doses and Results challenge CTAPV 1B CTAPV 1B:Challenge challenge T = 3 d p T = 7 d p T = 10 d p T = 14 d p load RNA T= 0 chall chall chall chall copies/ml Plasma Plasma Plasma Plasma Plasmain 10% RNA RNA RNA RNA RNA Animal Material Route homogenate copies/mlcopies/ml copies/ml copies/ml copies/ml 326 sentinels n.d. n.d. n.d.n.d. n.d. 365 n.d. n.d. n.d. n.d. n.d. 366 spleen + IM, 4 ml oral;6.15E+05 n.d. n.d. n.d. 2.38E+05 N/A 367 spinal nasal 2 x 1 ml IM n.d.n.d. n.d. 3.24E+04 2.00E+06 c + brain oral + 2 x 1 ml nasal; SC 2 x 1 mlSC 368 spleen + IM, 4 ml oraal; 8.65E+05 n.d. n.d. n.d. 3.50E+05 N/A 369tonsil + brain nasal 2 x 1 ml IM n.d. n.d. n.d. 2.16E+05 2.67E+06 oral +2 x 1 ml nasal; SC 2 x 1 ml SC 370 brain + IM, 4 ml oral; 3.91E+05 n.d.n.d. n.d. 3.24E+05 3.31E+06 371 spinal cord nasal 2 x 1 ml IM n.d. n.d.4.06E+04 5.23E+05 N/A oral + 2 x 1 ml nasal; SC 2 x 1 ml SC n.d.: notdetectable N/A: not analysed (animal already sacrificed)

In group B, six pigs were inoculated with CTAPV 8 homogenates via theintramuscular (IM), subcutaneous (SC), Intranasal (N) and oral (OR)routes.

Two pigs received inoculum from spleen+tonsil+brain+spinal cordhomogenate

Two pigs received inoculum from spleen+tonsil homogenate

Two pigs received inoculum from brain+spinal cord homogenate

Two pigs served as contact sentinels.

IM, SC and N volumes were 2.0 ml per dose, left and right. OR volume was3 or 4 ml. Nasal dose was sprayed. Challenge doses are given in Table 8.

After inoculation, all pigs were observed daily for clinical signs, butthe animals remained asymptomatic during the course of the experiment.

CPDA-blood, nose swabs, oropharynx swabs and rectal swabs were taken onday 0, day 3, day 7 and day 14 after inoculation to monitor infectionand excretion of CTAPV-8 via qPCR analysis. Plasma was obtained fromCPDA blood using the Leucosep® kit (Greiner Mat. no. 163 288). Theresults of qPCR analysis on plasma samples are presented in Table 8.

All inoculated animals showed a positive CTAPV qPCR result in bloodplasma at day 3 and/or day 7. Based on excretion of virus, animals weresacrificed at different time points to obtain fresh infectious materialfor subsequent in vitro and in vivo studies.

At the time of necropsy, brain, spinal cord, spleen, tonsils, and bloodwere taken from the animals.

The organ materials were used as challenge material in thevaccination-challenge study as described in Example 8/9

TABLE 8 Challenge doses and Results challenge CTAPV 8 CTAPV 8: Challengechallenge T = 3 d p T = 7 d p T = 14 d p load RNA T = 0 chall challchall copies/ml in Plasma Plasma Plasma Plasma 10% RNA RNA RNA RNAAnimal Material Route homogenate copies/ml copies/ml copies/ml copies/ml394 sentinels n.d. n.d. n.d. n.d. 395 n.d. n.d. n.d. n.d. 397 mix 4 IM,3 ml oral; 1.04E+06 n.d. 5.50E+03 2.55E+06 N/A 398 organs nasal 2 x 2 mlIM n.d. 5.22E+03 8.35E+04 N/A oral + 2 x 2 ml nasal; SC 2 x 2 ml SC 399spleen + IM, 4 ml oral; 1.03E+06 n.d. 7.92E+03 N/A N/A 400 tonsil nasal2 x 2 ml IM n.d. 2.46E+03 1.57E+05 N/A oral + 2 x 2 ml nasal; SC 2 x 2ml SC 401 brain + IM, 4 ml oral; 4.02E+05 n.d. 3.28E+03 1.73E+04 N/A 402spinal nasal 2 x 2 ml IM n.d. 5.07E+03 4.77E+06 N/A cord oral + 2 x 2 mlnasal; SC 2 x 2 ml SC n.d.: not detectable N/A: not analysed (animalalready sacrificed)

Example 7

Preparation of Challenge Material for Vaccination-Challenge Experiment

Challenge material was obtained from Example 6.

CTAPV 1B

Brains, spinal cord, spleen and tonsils of 1 necropsied animal ofexample 6, group A

CTAPV 8

Brains, spinal cord, spleen and tonsils of 1 necropsied animal ofexample 6, group B Pooled tissues were weighted after thawing.Subsequently, 9 times tissue-weight M6B8 medium with 10 μM HEPES (SigmaH3375-250G) was added to the tissue material. The tissue was homogenizedusing a blender, followed by shaking with small glass beads for 5minutes. During homogenizing organ-pulp was kept on ice. The organ-pulpwas centrifuged 1 hour at 3200×g. Supernatant was first passed over a0.45 μm filter, and subsequently trough a 0.22 μm filter with exceptionof the material for oral administration. The filtered homogenate wasstored at −70° C. until use.

Example 8

Vaccination-Challenge Experiment

Vaccine Design: Expression of E2 Protein:

The amino acids sequence of CTAPV 1 virus was analyzed. The start andstop of the E2 gene were determined using an alignment of the CTAPVvirus genome with Classical Swine Fever virus (CSF) E2 protein (Genbank:AAS 20412.1) and Bovine Virus Diarrhea virus (BVDV) E2 protein (Genbank:AGN03787.1), and predicted cleavage sites of the E2 protein weredetermined using SignalP4.1 software (www.cbs.dtu.dk/services/SignalP/)

The predicted amino acid sequence of CTAPV 1 E2 (SEQ ID NO: 32):

SCHKRQDYYSIQLVVDGKTGVEKRSIVGKWTVITREGREPRLMEQISMVSNDSLSETYCYNRLNTSSWGRQPARQRGCGQTVPFWPGDNVLEEQYYSTGYWVNATGGCQLREGVWLSRKGNVQCQRNGSSLILQLAIKEENDTMEIPCDPVETESMGPVTQGTCVYSWAFAPRGWYYNRKDGYWLQYVKKNDYQYWTKMP TASSATTMYRH

Subsequently, the CTAPV E2 nucleotide sequence for expression of CTAPVE2 protein in the Baculovirus expression system in insect cells wasoptimized using the Genscript OptimumGene™ algorithm (www.genscript.com)(SEQ ID NO: 33).

CGCGGATCCAAATATGTCATGTCACAAGCGTCAAGACTACTACTCTATCCAACTGGTGGTGGACGGAAAAACTGGCGTGGAAAAGCGTTCTATCGTGGGCAAGTGGACGGTCATCACCAGGGAGGGCAGAGAACCGCGCCTAATGGAGCAAATTTCGATGGTATCTAACGACTCTCTTTCAGAAACCTACTGCTATAACCGTCTCAATACTAGCTCTTGGGGTCGTCAACCTGCCCGTCAGCGCGGATGTGGGCAAACCGTCCCCTTCTGGCCTGGTGACAACGTACTCGAGGAACAGTACTATAGCACCGGATACTGGGTTAACGCTACTGGCGGTTGCCAACTACGCGAGGGAGTTTGGTTATCTCGTAAGGGGAACGTGCAATGTCAGCGTAATGGCTCATCGCTGATCCTTCAACTCGCTATTAAAGAGGAAAACGACACCATGGAAATCCCGTGCGATCCAGTCGAGACTGAATCAATGGGCCCCGTTACTCAAGGCACGTGTGTGTACAGCTGGGCTTTCGCCCCTAGGGGATGGTACTATAACCGTAAGGACGGCTACTGGCTTCAATACGTGAAGAAAAACGATTACCAGTACTGGACCAAAATGCCCACTGCATCCAGCGCGACCACTATGTACCGTCACC ATCACCATCACCATCACTAAGAATTCTCGAG

The restriction sites BamHI and EcoRI are underlined. The start codon isindicated in Italic and the stop codon is indicated in bold.

Transformation and Expression:

The E2 gene of CTAPV was synthesized at Genscript and directly cloned ina plasmid vector (pFastbac1) using the BamHI and EcoRI restrictionsites. The plasmid was transformed to E. coli using standardtransformation techniques, and subsequently plasmid DNA was purified andused for transfection of SF9 insect cells. The transfection was carriedout as follows:

2 ml cell suspension of 5*10⁵ cells/ml was added to each well of a 6well plate. The cells were allowed to attach to the plate for 1 hour at27° C. The following transfection solution (200 μl medium withoutantibiotics, 5 μl miniprep DNA and 6 μl cellfectin (Invitrogen)) wasprepared and incubated at room temperature for 45 minutes. After 45minutes 0.8 ml medium was added to the transfection solution and thiswas added to the attached cells. The transfected cells were incubatedfor 4 hours at 27° C. After 5 hours another 1 ml of medium (supplementedwith gentamycin and natamycin) was added to the cells. Cells were grownfor 3 days at 27° C. The supernatant was stored at −70° C. as P1 virusstock.

The expression of the CTAPV E2 protein in the SF9 cultures was checkedby SDS-page gel electrophoresis. The obtained samples from the SF9cultures were diluted 1:1 with Bio-Rad Laemmli sample buffer with 5%P3-mercaptoethanol, and subsequently samples were heated to 99° C. for10 minutes. All samples and a Precision Plus Protein™ All Blue (Bio-Rad)marker were loaded into a Bio-Rad CriterionMTGX™ precast gel (any kD™)and electrophoresed at 200 V for 42 minutes. The electrophoresis bufferused was 1× Tris/Glycine/SDS. After electrophoresis, the gel was stainedfor 1 hour in InstantBlue™ (Expedeon) protein staining buffer.

Purification:

After expression in SF9 cells, the E2 protein was purified in twodifferent ways. The first purification method was by making a whole celllysate. A SF9 culture expressing E2 of CTAPV was pelleted, resuspendedin PBS and sonicated using a Branson sonifier (2 times 30 pulses, output5, duty cycle 55%). After sonication the lysate was centrifuged for 10minutes at 8,000 rpm. The pellet containing the overexpressed E2 wasresuspended in PBS. Another way of purifying the E2 protein was by apurification method using IMAC and anionic detergents. This method isdescribed in BMC Biotechnology 2012, 12:95. (BMC Biotechnology 2012,12:95; Use of anionic denaturing detergents to purify insoluble proteinsafter overexpression; Benjamin Schlager, Anna Straessle and ErnstHafen). A lysis buffer containing an anionic denaturing detergent (SDS)was used to lyse the overexpressed E2 culture. The excess of detergentwas removed by cooling and purification, prior to affinity purification.

E2 proteins expressed in SF9 cells and purified as describe above wererun on SDS-page gel together with Bovine Serum Albumin standards withknown protein concentration. Protein concentration was estimated bycomparison of band intensities using Genetools software (Syngene version3.08.07).

Formulation

The final vaccine was formulated in a water-in-oil emulsion based onmineral oil. The water: oil ratio based on weight was 45:55. Dropletsize of the emulsion was mainly smaller than 1 m and viscosity was about80-150 mPa·sec.

Vaccine 1: water phase consisted of purified E2 protein (estimated E2concentration 60 μg/ml)

Vaccine 2: water phase consisted of whole cell lysate (estimated E2concentration 62 μg/ml)

Vaccination-Booster

For this experiment, 48 weaner-aged piglets at 5 weeks of age wereavailable. 3×8 animals per group were housed in stable 1, and 3×8animals per group were housed in stable 2. No contact between animalswas possible between stables.

Per group of 8 animals, 6 piglets receive a primo vaccination withvaccine 1, the other 2 piglets were not vaccinated at the beginning ofthe study.

At t=21 days, 5 out of 6 primo-vaccinated animals in each of the groupsreceived a booster vaccination with vaccine 2.

Blood samples were collected prior to primo vaccination, at day 21 afterinfection prior to booster vaccination, and at day 39, prior tochallenge

Of each group, 4 animals that received primo and booster vaccination,plus 2 non-vaccinated animals were moved to the challenge facilitiesprior to challenge.

Of each group, 1 animal that received only primo vaccination, and 1animal that received both primo and booster vaccination were monitoredfor an additional two weeks.

Challenge

The 36 animals for this experiment were housed in stable 3, 3×6 animalsper group, group 1-3, and in stable 4, 3×6 animals per group, group 4-6,were housed. The animals in stable 3 originated form stable 1, theanimals in stable 4 originated from stable 2.

No contact between animals was possible between stables. No physicalcontact was possible between animals of different groups within astable, but air-contact was possible.

Animals were challenged with live virus material on day 39 after primovaccination.

In stable 3, 3×6 piglets (group 1-3) were challenged with CTAPV 1challenge material (see above).

Group 1: 10.0 ml oral and 2×2.0 ml nasal

Group 2: 2×1.0 ml IM

Group 3: 2×1.0 ml IM

In stable 4 3×6 piglets (group 4-6) were challenged with CTAPV 8challenge material (see above).

Group 1: 10.0 ml oral and 2×2.0 ml nasal

Group 2: 2×1.0 ml IM

Group 3: 2×1.0 ml IM

Serum blood samples and nasal, rectal and oropharynx swabs werecollected prior to challenge, and at 3, 6, 9, 13, 16, 20, 23 and 27 dayspost challenge to monitor infection and excretion of CTAPV viruses viaqPCR analysis. Three animals (two vaccinated, one non-vaccinated) pergroup were necropsied at day 13 post challenge, the other 3 animals (twovaccinated, one non-vaccinated) were necropsied at day 27 postchallenge. Inguinal lymph nodes, mesenteric lymph nodes and tonsils weresampled at the time of necropsy.

Example 9

Antibodies to CTAPV E2 Protein

Expression of E2 Protein in E. Coli:

The amino acids sequence of CTAPV 1 virus was analyzed. The start andstop of the E2 gene were determined using an alignment of the CTAPVvirus genome with Classical Swine Fever virus (CSF) E2 protein (Genbank:AAS 20412.1) and Bovine Virus Diarrhea virus (BVDV) E2 protein (Genbank:AGN03787.1), and predicted cleavage sites of the E2 protein weredetermined using SignalP4.1 software (www.cbs.dtu.dk/services/SignalP/)

The predicted amino acid sequence of CTAPV 1 E2 (SEQ ID NO: 32):

SCHKRQDYYSIQLVVDGKTGVEKRSIVGKWTVITREGREPRLMEQISMVSNDSLSETYCYNRLNTSSWGRQPARQRGCGQTVPFWPGDNVLEEQYYSTGYWVNATGGCQLREGVWLSRKGNVQCQRNGSSLILQLAIKEENDTMEIPCDPVETESMGPVTQGTCVYSWAFAPRGWYYNRKDGYWLQYVKKNDYQYWTKMP TASSATTMYRHProtein Sequence for Expression in E. Coli (Includes a HIS-Tag)

SCHKRQDYYSIQLVVDGKTGVEKRSIVGKWTVITREGREPRLMEQISMVSNDSLSETYCYNRLNTSSWGRQPARQRGCGQTVPFWPGDNVLEEQYYSTGYWVNATGGCQLREGVWLSRKGNVQCQRNGSSLILQLAIKEENDTMEIPCDPVETESMGPVTQGTCVYSWAFAPRGWYYNRKDGYWLQYVKKNDYQYWTKMP TASSATTMYRHHHHHHH

Subsequently, the CTAPV E2 nucleotide sequence for expression of CTAPVE2 protein in E. Coli was optimized using the Genscript OptimumGene™algorithm (www.genscript.com) (SEQ ID NO: 34).

CATATGTCGTGTCACAAACGCCAAGATTATTATTCTATTCAACTGGTCGTGGATGGTAAAACGGGTGTCGAAAAACGCTCTATCGTCGGTAAATGGACCGTGATTACGCGTGAAGGCCGCGAACCGCGTCTGATGGAACAGATCAGTATGGTTTCCAACGATAGCCTGTCTGAAACCTATTGCTACAACCGCCTGAATACGAGCTCTTGGGGTCGTCAGCCGGCACGTCAACGCGGCTGTGGTCAGACCGTCCCGTTTTGGCCGGGCGACAACGTGCTGGAAGAACAATATTACAGTACCGGTTATTGGGTGAATGCAACGGGCGGTTGCCAGCTGCGTGAAGGCGTTTGGCTGTCTCGTAAGGGTAACGTCCAGTGTCAACGCAATGGCAGTTCCCTGATTCTGCAACTGGCGATCAAAGAAGAAAACGATACCATGGAAATCCCGTGCGACCCGGTCGAAACCGAATCAATGGGCCCGGTGACCCAGGGCACGTGTGTTTATTCGTGGGCATTCGCACCGCGCGGCTGGTATTACAACCGTAAAGATGGTTATTGGCTGCAGTACGTGAAGAAAAACGACTATCAATACTGGACCAAAATGCCGACGGCATCATCGGCTACCACGATGTACCGTCATCACCATCACCA TCACCATTAACTCGAG

Restriction sites added (in bold) are NdeI and XhoI.

Transformation and Expression:

The E2 gene of CTAPV was synthesized at Genscript and directly cloned ina plasmid vector (pET22b) using the NdeI and XhoI restriction sites. Theplasmid was transformed to E. coli BL21star+pLysS using standardtransformation techniques, and expression was induced.

Expression was achieved by growing the expression strains inautoinducing media for 18 hours at 37° C.

Expression was verified by running SDS-page gel electrophoresis.

E2 was found to be in the insoluble fraction. The E2 protein waspurified by applying a purification method using IMAC and anionicdetergents. This method is described in BMC Biotechnology 2012, 12:95.(BMC Biotechnology 2012, 12:95; Use of anionic denaturing detergents topurify insoluble proteins after overexpression; Benjamin Schlager, AnnaStraessle and Ernst Hafen). A lysis buffer containing an anionicdenaturing detergent (SDS) was used to lyse the overexpressed E2culture. The excess of detergent was removed by cooling andpurification, prior to affinity purification.

The purified protein was checked on SDS-page as described in Example 8.The purified protein was formulated in GNE and used for injection ofrabbits to generate antibodies. The estimated concentration of theprotein in the water phase was 0.5 mg/ml.

FIG. 7 shows that the antibodies raised in rabbits (serum t=4 weeksafter vaccination) specifically recognizes an approximately 25 kDa bandthat corresponds to the CTAPV E2 protein expressed in thebaculovirus/SF9 expression system (lane 2). Lane 1 contains a marker andLane 3 contains an unrelated expression product in the baculovirus/SF9expression system.

Example 10

SYBR Green One-Step qRT-PCR

Animal Samples

Swine serum and spleen samples were collected from experimentallyinfected and control pigs. Blood was collected (Vacuolette 8 ml Sep ClotActivator ref: 455071; Greiner Bio-one) and serum was obtained bycentrifugation 20 minutes at 3,000×g at 4° C. Sperm samples wereobtained from a commercial breeding company and tested withoutpretreatment.

10% Tissue homogenates were prepared in PBS on ice. Homogenization wasperformed in Gentle Macs M tubes with the Gentle Macs Dissociator(Miltenyi Biotec). This homogenized material was then centrifuged twice,first at 3,200×g for 30 minutes and subsequently at 10,000×g for 10minutes. Subsequently a DNase treatment was done: 24 μl 10× Turbo DNasebuffer and 20 μl Turbo DNase (AMbion) was added to 250 μl supernatantand this mixture was incubated at 37° C. for 10 minutes.

RNA Extraction

RNA was extracted from these samples with the Magnapure 96 instrument(Roche) with external lysis. This system purifies DNA, RNA, and viralnucleic acids using magnetic glass particle technology. 200 μl samplewas mixed with 250 μl magnapure total nucleic acid isolation kitlysis/binding buffer and the extraction was performed in the Magnapureinstrument using the external lysis protocol. RNA samples were stored at−70° C. until further use.

SYBR Green One-Step qRT-PCR

Specific Primer Design

Oligonucleotide primers were used to amplify the 5′ UTR genome of theCTAPV genome. This part of the viral genome was chosen based onconserved nucleotide sequence between CTAPV variants 1-9 (based onalignment of the nucleotide sequences). The primer sequences were asfollows: CTAPV-PAN2-F3-B: CGTGCCCAAAGAGAAATCGG (SEQ ID NO: 35) andCTAPV-PAN2-R3-B (SEQ ID NO: 36): CCGGCACTCTATCAAGCAGT.

qRT-PCR Protocol

A SYBR green based one step qRT-PCR was developed using the SuperscriptIII Platinum SYBR Green One-Step qRT-PCR kit (ThermoFisher). Eachreaction contained 25 μl 2×SYBR Green Reaction Mix, 1 μl Superscript IIIRT/Platinum Taq Mix, 1 μl 10 μM CTAPV-PAN2-F3-B primer, 1 μl 10 μMCTAPV-PAN2-R3-B primer, 17 μl RNAse free water and 5 μl RNA template.All reaction were performed on a BioRad CFX96 with the following cyclingparameters; a RT reaction at 55° C. for 3 min, Pre-denaturation at 95°C. for 5 min and then 40 cycles of 95° C. for 15 sec, 60° C. for 30 secfollowed by a melting curve program from 60° C. until 95° C. with 0.5°C./5 sec.

Standard Line Creation

For quantification of the detected RNA in the SYBR Green One-StepqRT-PCR a standard line was constructed containing the q-PCR targetsequence of which standard dilutions can be calculated. A 177 base pairslong sequence from the 5′UTR part (162-338) of the CTAPV genome wassynthesized (Genscript) and ligated in a pUC57 vector that wassubsequently transfected in E. coli. Plasmid DNA was isolated bymidiprep.

The formula for calculating plasmids copies/μl is:Plasmid copies/μl=DNAconcentration(ng/μl)/((A×328.4+G×344.24+T×303.22+C×304.16)/(6.02×10²³))×2×10⁹).

The DNA concentration of the plasmid was 100 ng/l. Eight dilutions weremade containing 10⁸ until 10¹ copies/2 μl.

Results

Validation of the qRT-PCR.

A standard line with eight dilutions containing 10⁸ until 10¹ copies/5μl and a negative control sample were included in an experiment tovalidate the qRT-PCR. FIG. 1A shows a diagram in which the qPCR cycliare plotted against the relative fluorescence units in real time. Eachsample was tested in duplicate. The straight line at about 100 RFU isthe cut-off line, the straight line at 0 RFU is the negative controlsample. The duplicate sample with the highest quantity of template isthe sample that shows the initial fluorescence increase around cycle 10(10⁸, followed by 10⁷ at cycle 12 etc). FIG. 1B was prepared from thesame experimental data, but here the Log starting quantity standardcurve (o) is plotted against the quantification cycle. The standard linehas an efficiency of 102% and a R² of 0.997, this is within the rangefor a specific and quantifiable qPCR in which the efficiency should bebetween 95% and 105% and the R² must be above 0.990. FIG. 1C shows themelting curves of the samples shown in panels A and B. All positivesamples show identical curves and a specific melting point, which meansa specific fragment is amplified and that the fragment is identical ineach of the reactions.

These data show that the developed qRT-PCR meets the requirements forthe detection and quantification of CTAPV. The qRT-PCR was subsequentlyused for sample analysis of suspected CTAPV positive samples and controlsamples. Interpretation of the data was based on the RFU per cycle plusthe characteristics of the melting curve. Aberrant melting curves wouldbe indicative for non-specificity of the amplicon.

Detection and Quantification of CTAPV RNA in Serum, Spleen and SpermSamples.

Serum and spleen from experimentally infected and from control giltswere tested in duplicate for CTAPV RNA presence. Also, sperm sampleswere tested. The (average) results are presented in Table 9. In theassays performed, the standard lines were confirmed to be within thequality range for an accurate qPCR (FIG. 9B, see above). Also, the CTAPVspecific melting point was confirmed in the melting curves of all thesesamples. Based on these data, we can conclude that the qRT-PCR isappropriate for the detection and quantification of CTAPV RNA in serum,spleen and sperm samples.

TABLE 9 CTAPV RNA quantification of swine serum, sperm and spleensamples. Ct values* RNA copies/5 μl* RNA copies/ml* Serum CTAPV 27.419.99E+02 5.00E+04 positive gilt 1 Serum CTAPV ND — — negative gilt 2Spleen CTAPV 29.45 7.93E+02 3.97E+04 positive gilt 3 Spleen CTAPV ND — —negative gilt 4 Sperm CTAPV 29.75 5.39E+02 2.70E+04 positive boar 1Sperm CTAPV ND — — negative boar 2 *Means of duplicate experiments; ND:not detectable; spleen refers to 10% (w/v) homogenate sample. Column RNAcopies/5 μl shows the number of copies of the virus in 5 μl extractedRNA sample obtained from 200 μl of the original sample. Column RNAcopies/mL shows the number of copies of the virus in the original sample(serum, sperm) or the 10% homogenate (spleen).

Example 11

CTAPV Positive Sperm Infects Gilts and Offspring

Animals

Six gilts were obtained from an SPF/High Health farm. Sperm from aCTAPV-positive boar was used for artificial insemination of the gilts.

Methods

Blood was collected from gilts and offspring (Vacuolette 5/8 ml Sep ClotActivator ref: 455071; Greiner Bio-one) and serum was obtained bycentrifugation 20 minutes at 3,000×g at 4° C. Sperm samples were testedwithout pretreatment.

RNA extraction and qRT-PCR were performed as described in the section“SYBR Green One-Step qRT-PCR” of Example 10.

Results

Tested gilts were serum-negative for CTAPV prior to insemination(qRT-PCR). Boar sperm was positive for CTAPV as analysed by qRT-PCR. Att=+4 weeks after insemination, gilts 4 and 5 contained detectable levelsof CTAPV in serum. At the day of farrowing, gilts 1, 2 and 6 containeddetectable levels of CTAPV in serum. Piglets with detectable levels ofCTAPV in serum were born out of 5 of 6 gilts (see Table 10 for results).Piglets were healthy and showed no clinical tremor or increasedincidence of other clinical symptoms related to congenital tremor typeAII such as splay legs.

TABLE 10 CTAPV positive sperm infects gilts and offspring RNA copies t =4 w RNA copies/mL Results qRT-PCR serum gilt gestation at farrowing*piglets: clinical score: 52 1.65E+02 6 out of 10 10× no CTAPV positivecongenital tremor 53 4.15E+01 6 out of 11 11× no CTAPV positivecongenital tremor 54  ND** 0 out of 16 16× no CTAPV positive congenitaltremor 55 2.47E+02 ND 1 out of 15 15× no CTAPV positive congenitaltremor 56 8.70E+01 ND 3 out of 17 17× no CTAPV positive congenitaltremor 57 ND 3.19E+04 11 out of 18 18× no CTAPV positive congenitaltremor *Column RNA copies shows the number of RNA copies of the virusper mL in the original sample (serum) **ND: not detected/below detectionlevel

Example 12

Infection of Pregnant Gilts with CTAPV Variant 1B Obtained from “ShakingPiglets” and Effect on Newborn Piglets: CTAPV Positive Sperm

Animals

Six gilts were obtained from a SPF/high health farm. Gilts wereinseminated via artificial insemination with CTAPV positive sperm.Pregnancy was confirmed at day 28 of gestation using ultrasound. Allgilts gave birth to a litter of piglets on day 115 or day 116 ofgestation.

Infection

Three of the gilts were infected on day 32 after insemination with aCTAPV1B inoculum consisting of organ homogenates of spleen and brainobtained from necropsied pig 371 at t=11 days after infection withCTAPV1B infected material. This experiment was described in Example6/Table 7. The homogenate was prepared as follows. To 14 grams of spleenand 8 grams of brain, 9 times tissue-weight M6B8 medium (MSD AH) with 10μM HEPES (Sigma H3375-250G) was added. The tissue was homogenized usinga blender, followed by shaking with small glass beads for 5 minutes.During homogenizing and subsequent processing, organ-pulp was kept onice. The organ-pulp was centrifuged 1 hour at 3200×g at 4° C.Supernatant was passed over a 0.22 μm filter. The filtered homogenatewas stored at −70° C. until use. These three gilts received anintramuscular injection of 5 mL inoculum (two injections of 2.5 mL eachin the left and right neck).

The other three gilts were infected with an inoculum of serum obtainedfrom the same pig at the time of necropsy. The serum was filtered over a0.22 μm filter prior to injection. These three gilts received anintramuscular injection of 5 mL inoculum (two injections of 2.5 mL eachin the left and right neck).

The quantitative amount of CTAPV in the inoculums was determined byqRT-PCR as described in Example 10.

Serum Collection

Serum was collected prior to infection of the gilts, and at t=10 daysafter insemination. Serum was also collected from newborn piglets withinhours after birth. Blood was collected (Vacuolette 5/8 ml Sep ClotActivator ref: 455071; Greiner Bio-one) and serum was obtained bycentrifugation 20 minutes at 3,000×g at 4° C. RNA extraction and qRT-PCRwere performed as described in the section “SYBR Green One-StepqRT-PCR”, Example 10.

Results

The mixed homogenate of spleen and brain used for infection of the firstthree gilts contained 4.5 E+02 genomes copies per 5 μl of the extractedRNA. This equals 2.3 E+04 genome copies per mL in the homogenate thatwas used for infection of the gilts.

The serum inoculum used for infection of the other three gilts contained1.2 E+04 genomes per 5 μl of the extracted RNA, which equals 6.0E+05genome copies per mL that was used for infection of gilts. Table 11presents the quantitative amount (genomes per mL serum) at day 10 postinfection as determined by qRT-PCR results. Five out of six gilts gavebirth to piglets with severe congenital tremor type A-II. One gilt, thegilt with a relatively low virus quantity in the serum at t=10 daysafter infection, gave birth to a relatively healthy litter where only 2piglets with mild symptoms were observed. Litter information scoredafter farrowing is presented in Table 11. An increased incidence ofsplay legs was associated with clinical tremor, as described by M. White(www.nadis.org.uk/bulletins/congenital-tremor.aspx?altTemplate=PDF).

Presence of CTAPV in three piglets per litter (those with severeclinical tremor, except those piglets born from gilt 44 which showed noclinical tremor) was tested by the qRT-PCR test described in Example 10.The number of CTAPV positive piglets is depicted in Table 11

TABLE 11 RNA quantitation in gilt serum samples on day 10 afterinoculation, and litter information. CT type A-II piglets CTAPV RNA born(live piglets- presence in piglets copies/ # severe/# mild/# (pigletstested/ Gilt Infection mL* no symptoms) piglets positive) 42 Organ1.1E+05  9-4/4/1 3/3 homogenate 43 Organ 4.5E+04 15-7/7/1 3/3 homogenate44 Organ 5.5E+02  14-0/2/12 3/2 homogenate 45 Serum 1.2E+05 18-9/8/0 3/3(1 not scored) 47 Serum 1.3E+05  16-11/4/1 3/3 48 Serum 1.2E+05 15-8/7/03/3 *Means of duplicate experiments; Column RNA copies/mL shows thenumber of copies of the virus in the original sample (serum).

Example 13

Infection of Pregnant Gilts with CTAPV Variant 1B Obtained from “ShakingPiglets” and Effect on Newborn Piglets: CTAPV Negative Sperm

Animals

Three gilts were obtained from a SPF/high health farm. Gilts wereinseminated via artificial insemination with CTAPV negativeregnancy wasconfirmed at day 28 of gestation using ultrasound. All gilts gave birthto a litter of piglets on day 114 or day 115 of gestation.

Infection

The three gilts were infected with an inoculum of serum obtained frompig 371 at the time of necropsy (see example 12). The serum was filteredover a 0.22 μm filter prior to injection. Three gilts received anintramuscular injection of 5 mL inoculum (two injections of 2.5 mL eachin the left and right neck) at 32 days of gestation.

The quantitative amount of CTAPV in the inoculum was determined byqRT-PCR as described in Examples 10 and 12.

Serum Collection

Serum was collected prior to infection of the gilts, and at t=10 daysafter insemination. Serum was also collected from newborn piglets withinhours after birth. Blood was collected (Vacuolette 5/8 ml Sep ClotActivator ref: 455071; Greiner Bio-one) and serum was obtained bycentrifugation 20 minutes at 3,000×g at 4° C. RNA extraction and qRT-PCRwere performed as described in the section “SYBR Green One-StepqRT-PCR”, Example 10.

Results

Table 12 presents the quantitative amount (genomes per mL serum) at day10 post infection as determined by qRT-PCR results. Two of three giltsgave birth to piglets with mild congenital tremor type A-II. One gilt,the gilt with a relatively low virus quantity in the serum at t=10 daysafter infection, gave birth to a healthy litter. Litter informationscored after farrowing is presented in Table 11.

Presence of CTAPV in piglets with CT type A-II was confirmed by theqRT-PCR test described in Example 10. The number of CTAPV positivepiglets is depicted in Table 12. An increased incidence of splay legswas associated with clinical tremor.

TABLE 12 RNA quantitation in gilt serum samples on day 10 afterinoculation, and litter information. CT type A-II piglets CTAPV RNA born(live piglets -# presence in piglets copies/ severe/# mild/# (pigletstested/ Gilt Infection mL* no symptoms) piglets positive) 49 Serum5.85E+02 13-0/0/13 13/0  50 Serum 1.39E+04 13-3/8/2  13/11 51 Serum2.32E+04 15-1/12/2 15/15 *Means of duplicate experiments; Column RNAcopies/mL shows the number of copies of the virus in the original sample(serum).

The invention claimed is:
 1. A gene encoding an E2 protein produced byheterologous expression in a non-pestivirus expression system, whereinthe nucleotide sequence of said gene has a level of identity of at least80% to the nucleotide sequence of SEQ ID NO:
 3. 2. The gene of claim 1,wherein the non-pestivirus expression system is a baculovirus or yeastexpression system.
 3. A DNA fragment comprising the E2 gene of claim 1,wherein the E2 gene is under the control of a functional heterologouspromoter.
 4. A method of generating the E2 protein of claim 1,comprising expressing a gene encoding the E2 protein in a non-pestivirusexpression system, wherein the nucleotide sequence of said gene has alevel of identity of at least 80% to the nucleotide sequence of SEQ IDNO:
 3. 5. A method of generating the E2 protein of claim 1, comprisingincubating cells transfected with the non-pestivirus expression system,lysing the incubated cells, purification of the E2 protein from thelysed cells, and subsequent formulation of the purified E2 protein.